Registration Dossier

Diss Factsheets

Administrative data

Key value for chemical safety assessment

Toxic effect type:
concentration-driven

Effects on fertility

Description of key information

The available two-generation reproductive toxicity studies with the test substance in rats, indicate no concern for reproductive toxicity for the test substance.

Link to relevant study records

Referenceopen allclose all

Endpoint:
two-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1990
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
EPA OPP 83-4 (Reproduction and Fertility Effects)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
Test animals
- Source: Sprague-Dawley CD rats were obtained from Charles River Breeding Laboratories, Kingston, NY, USA
- Age at study initiation: Six weeks
- Weight at study initiation: 212.2-213.4 g (males); 148.3-150.2 g (females)
- Housing: Individually in stainless steel, wire mesh cages (22.5x15.5x18.0 cm; mating cages 22.5x31.0x18.0 cm)
- Diet: Certified Ground Rodent Chow # 5002 (Ralstor Purina Co., St. Louis, MO), ad libitum
- Water: Tap water, ad libitum. Water was provided by an automatic watering system with demand control valves mounted on each cage rack.
- Acclimation period: Two weeks

Environmental conditions
- Temperature: 66-73°F
- Humidity: 40-60%
Route of administration:
oral: feed
Vehicle:
other: Certified Ground Rodent Chow # 5002
Details on exposure:
Diet preparation: A concentrated premix was prepared by direct addition of the test substance to ground chow and mixing for approximately an hour. Test diets were prepared by appropriate dilutions of the concentrated premix or higher diet concentrations.
- Rate of preparation of diet (frequency): Fresh diet was prepared weekly.
- Mixing appropriate amounts with (Type of food): Certified Ground Rodent Chow # 5002
- Storage temperature of food: Diets were stored in polyethylene containers at room temperature.
Details on mating procedure:
- M/F ratio per cage: 1:1
- Mating procedure: After pre-breeding exposure the animals were mated on the basis of one male to one female selected randomly within each dose group for a period of 21d to produce F1 generation.
- Proof of pregnancy: Copulation plug and/or vaginal sperm as Day 0 of gestation.
- After the first 7d of the mating period females of unsuccessfully mated pairs were placed with males of other unmated pairs within the same dose group; after an additional 7d, unsuccessfully mated pairs were similarly exchanged again for a period of 7d or until successful mating had occurred, whichever came first, allowing for a total of 21d to mate.
- After successful mating each pregnant female was caged: Pregnant females were housed individually. On Day 20 of gestation each pregnant female was transferred to a shoe-box cage.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Experimental diets were analyzed for stability, homogeneity and concentration of test substance using high pressure liquid chromatography.
- Homogeneity study indicated that the distribution of the test substance in the test diet was uniform.
- Stability study indicated that the dosed feed was stable for at least 14d when stored in open glass feed jars at room temperature. Dosed feed was stable for at least 21d when stored in closed polyethylene containers at room temperature.
- Concentration verification analyses of the dosed feed indicated that the mean concentrations of the test substance in the diet for the 300, 1000 and 2000 ppm dosage levels were 95.3-109.0% of nominal for 300 ppm, 95.6-107.9% of nominal for 1000 ppm and 94.7-108.0% of nominal for 2000 ppm.
Duration of treatment / exposure:
P0 generation: 19 weeks (from 1st prebreed dose to last F0 sacrifice)
F1 generation: 25 weeks (from 1st F1 wean to last F1 sacrifice)
F2 generation: Until weaning
Frequency of treatment:
Daily
Details on study schedule:
- P1 parental animals not mated until 17-18 weeks after selection from the F1 litters.
- Selection of parents from F1 generation when pups were 28d old.
- Number of P1 generation animals selected: 28 males and 28 females

Remarks:
Doses / Concentrations:
0, 300, 1000 or 2000 ppm test substance (i.e., equivalent to 0, 16-31, 51-102, and 100-188 mg/kg bw/day (males) and 0, 21-32, 67-106 and 139-198 mg/kg bw/day (females)).
Basis: nominal in diet
No. of animals per sex per dose:
28
Control animals:
yes, plain diet
Details on study design:
- Rationale for animal assignment: Animals were randomly distributed based on body weight
- Animal identification: By ear tags
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily

CLINICAL SIGNS: Yes
- Time schedule: Once daily for overt clinical signs.

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly during prebreed and mating for both sexes. For females on gestational Day 0, 6, 15, and 20 and on postnatal Day 0, 7, 14, and 21.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule: Weekly during prebreed for both sexes. For females in 3- or 4-day intervals throughout gestation and to postnatal Day 14.
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

WATER CONSUMPTION AND COMPOUND INTAKE: No

Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: Yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible). Excess pups were subjected to detailed external examination and then sacrificed.

PARAMETERS EXAMINED
The following parameters were examined in F1 and F2 offspring: Live/Still births on the day of birth (postnatal Day 0), survival indices at Days 0, 4, 7 and 14 after birth and weaning, weight on postnatal Day 1, 4, 7, 14, and at weaning (Day 21), and physical abnormalities for all pups at birth and throughout the pre-weaning period.

GROSS EXAMINATION OF DEAD PUPS: The thoracic and abdominal organs from pups which died after Day 4 were preserved for subsequent histopathological examination.
Postmortem examinations (parental animals):
SACRIFICE: Yes
- How many animals: All animals
- Necropsy method: Animals were anesthetized with methoxyflurane and exsanguinated by severing the brachial vessel.

GROSS NECROPSY: Yes
- How many animals: All animals sacrificed in F0 and F1 parental animals
- Gross necropsy consisted of: Vagina, uterus, ovaries, gross lesions, testes, epididymis, seminal vesicles and prostrate.

HISTOPATHOLOGY : Yes
- How many animals: All animals of control and high dose groups sacrificed in P0 and P1 parental animals
- Tissues evaluated: Vagina, uterus, ovaries, gross lesions, testes, epididymis, seminal vesicles and prostrate and any tissues or organs showing gross alterations from the low and mid dose groups
Postmortem examinations (offspring):
SACRIFICE
- The F1 offsprings not selected as parental animals and all F2 offsprings were sacrificed at 7d of age.

GROSS NECROPSY
- Examination for gross lesions was performed on any pup appearing abnormal or dying on test and for ten randomly selected F1 and F2 weanlings/sex/group.

Statistics:
- The results of the quantitative continuous variables (e.g., body weights, food consumption, etc.) were compared between the three treatment groups and one control group using Levene’s test for equal variances, analysis of variance and (pooled or separate variance) t-test.
- Non-parametric data were statistically evaluated using the Kruskall-Wallis test followed by the Mann-Whitney U test for pairwise comparisons when appropriate.
- Frequency data were compared using the Fisher’s exact test.
Reproductive indices:
Mating index, fertility index and gestational index were determined.
Offspring viability indices:
Live birth index, 4-d survival index, 7-d survival index, 14-d survival index, 21-d survival index and lactation index

Clinical signs:
no effects observed
Description (incidence and severity):
No significant signs of toxicity during the pre-breed, mating, gestation or lactation periods at any dose for either generation were observed.



Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- During the 10-week pre-breed exposure, F0 males exhibited no reduction in body weight. During the same period, F0 females at 2000 ppm exhibited reduction in body weight during Weeks 5, 6, 9 and 10 of pre-breed treatment. Body weight gain was also reduced at 2000 ppm for one week (Week 8-9) during the pre-breed period.
- On lactation Day 21 mean body weight of F0 dams at 2000 ppm exhibited a significant increase. Increased lactation body weight gain was observed at 2000 ppm throughout lactation.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
- Food consumption in the F0 females at 2000 ppm was reduced for the first four exposure weeks. Food consumption in F0 males was significantly reduced at 2000 ppm for the first week of treatment only.
- At F0 breed to produce F1 litters, food consumption during gestation and lactation was unaltered by treatment.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Other effects:
effects observed, treatment-related
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Description (incidence and severity):
Mating index, fertility index and gestational index
No treatment-related effects on any reproductive parameters were observed at any dose; NOEL (parental) = 1000 ppm
Key result
Dose descriptor:
NOAEL
Remarks:
systemic toxicity
Effect level:
1 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
Remarks on result:
other: i.e., equivalent to 51-102 mg/kg bw/day (or 41-83 mg a.i./kg bw/day) in males and 67-106 mg/kg bw/day (or 54-86 mg a.i./kg bw/day) in females
Key result
Dose descriptor:
NOEL
Remarks:
reproductive toxicity
Effect level:
2 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no reproductive toxicity observed
Remarks on result:
not determinable due to absence of adverse toxic effects
Remarks:
i.e., equivalent to 100-188 mg/kg bw/day (or 81-152 mg a.i./kg bw/day) in males and 139-198 mg/kg bw/day (or 113-161 mg a.i./kg bw/day) in females
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
1 000 ppm
System:
other: body weight and food consumption
Treatment related:
yes
Dose response relationship:
yes
Clinical signs:
no effects observed
Description (incidence and severity):
No significant signs of toxicity during the pre-breed, mating, gestation or lactation periods at any dose for either generation were observed.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 2000 ppm, only slight reduction were observed in males and females
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Description (incidence and severity):
Mating index, fertility index and gestational index
Key result
Dose descriptor:
NOAEL
Remarks:
systemic toxicity
Effect level:
1 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
Remarks on result:
other: i.e., equivalent to 51-102 mg/kg bw/day (or 41-83 mg a.i./kg bw/day) in males and 67-106 mg/kg bw/day (or 54-86 mg a.i./kg bw/day) in females
Key result
Dose descriptor:
NOEL
Remarks:
reproductive toxicity
Effect level:
2 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no reproductive toxicity observed
Remarks on result:
not determinable due to absence of adverse toxic effects
Remarks:
i.e., equivalent to 100-188 mg/kg bw/day (or 81-152 mg a.i./kg bw/day) in males and 139-198 mg/kg bw/day (or 113-161 mg a.i./kg bw/day) in females
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
2 000 ppm
System:
other: body weight
Treatment related:
yes
Dose response relationship:
yes
Clinical signs:
no effects observed
Description (incidence and severity):
There were no signs of toxicity in the F1 or F2 animals.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Description (incidence and severity):
There was no effects of treatment on postnatal deaths.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The F1 litters exhibited reduced body weight per litter on postnatal Days 21 and 28 at 2000 ppm. F1 pup body weight gain was reduced during lactation Days 14-21 and 21-28.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no treatment-related observations or histopathological findings in either the F0 or F1 adult animals at any dose. There were no treatment-related findings in the F1 or F2 pups that died during lactation or randomly selected F1and F2 pups at necropsy.
Histopathological findings:
no effects observed
Description (incidence and severity):
There were no treatment-related observations or histopathological findings in either the F0 or F1 adult animals at any dose. There were no treatment-related findings in the F1 or F2 pups that died during lactation or randomly selected F1and F2 pups at necropsy.
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
NOEL (F1and F2 offspring) = 1000 ppm
Key result
Dose descriptor:
NOAEL
Remarks:
general toxicity
Generation:
F1
Effect level:
1 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
Remarks on result:
other: i.e., equivalent to 51-102 mg/kg bw/day (or 41-83 mg a.i./kg bw/day) in males and 67-106 mg/kg bw/day (or 54-86 mg a.i./kg bw/day) in females
Key result
Dose descriptor:
NOEL
Remarks:
developmental toxicity
Generation:
F1
Effect level:
2 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no effects observed
Remarks on result:
other: i.e., equivalent to 100-188 mg/kg bw/day (or 81-152 mg a.i./kg bw/day) in males and 139-198 mg/kg bw/day (or 113-161 mg a.i./kg bw/day) in females
Key result
Critical effects observed:
no
Clinical signs:
no effects observed
Description (incidence and severity):
There were no signs of toxicity in the F1 or F2 animals.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Description (incidence and severity):
There was no effects of treatment on postnatal deaths.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
F2 pup weights per litter were reduced at 2000 ppm on postnatal Day 28. Pup weight gain was also reduced at 2000 ppm during lactation Days 14-21 and for Days 21-28.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no treatment-related observations or histopathological findings in either the F0 or F1 adult animals at any dose. There were no treatment-related findings in the F1 or F2 pups that died during lactation or randomly selected F1and F2 pups at necropsy.
Histopathological findings:
no effects observed
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
NOEL (F1and F2 offspring) = 1000 ppm
Key result
Dose descriptor:
NOAEL
Remarks:
general toxicity
Generation:
F2
Effect level:
1 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
Remarks on result:
other: i.e., equivalent to 51-102 mg/kg bw/day (or 41-83 mg a.i./kg bw/day) in males and 67-106 mg/kg bw/day (or 54-86 mg a.i./kg bw/day) in females
Key result
Dose descriptor:
NOEL
Remarks:
developmental toxicity
Generation:
F2
Effect level:
2 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no effects observed
Remarks on result:
other: i.e., equivalent to 100-188 mg/kg bw/day (or 81-152 mg a.i./kg bw/day) in males and 139-198 mg/kg bw/day (or 113-161 mg a.i./kg bw/day) in females
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
no

For result tables, kindly refer to the attached background material section of the IUCLID.

Conclusions:
Under the study conditions, the rat NOAEL (systemic toxicity) for both parental generation (P0 and F1) and offspring (F1 and F2) was considered to be at 1000 ppm (in diet), i.e., equivalent to 51-102 mg/kg bw/day (or 41-83 mg a.i./kg bw/day) in males and 67-106 mg/kg bw/day (or 54-86 mg a.i./kg bw/day) in females, respectively. The rat NOEL (reproductive toxicity) although not specified by the authors in the study report, can be considered to be at the highest tested dose 2000 ppm (in diet), i.e., equivalent to 100-188 mg/kg bw/day (or 81-152 mg a.i./kg bw/day) in males and 139-198 mg/kg bw/day (or 113-161 mg a.i./kg bw/day) in females.
Executive summary:

A two-generation study was conducted to determine the toxicity to reproduction of the test substance, C12-16 ADBAC (81.09% active in aqueous/ethanol solution) according to US EPA OPP 83-4, in compliance with GLP. The test substance was orally administered to Sprague-Dawley CD rats (28/sex/group) at dose levels of 0, 300, 1000 or 2000 ppm test substance (equivalent to average doses of 0, 16-31, 51-102, and 100-188 mg/kg bw/day for males and 0, 21-32, 67-106 and 139-198 mg/kg bw/day in females) in the diet. Following a 10-week pre-breed exposure period, the P0 rats were randomly paired within dose groups for a 3-week mating period to produce the F1 generation. Exposure continued through mating, gestation, parturition and lactation. At weaning, twenty-eight (28) F1 weanling/sex/group were randomly selected and exposed to the same dietary concentration of the test substance as their parents for 10 weeks. After their pre-breed exposure, F1/P1 animals were paired to produce the F2 generation. The animals were observed twice daily and clinical findings, body weights and food consumption were recorded periodically. All P0 and F1 animals were necropsied and examined for gross lesions. Selected reproductive tissues from the high dose and control groups were examined histologically as were other tissues with gross lesions. Ten F1 and F2 weanlings/sex/dose were randomly selected and necropsied and examined for gross lesions. Remaining non-selected F1 and F2 pups at weaning were euthanised and discarded. Reduced body weights in F0 males (but no F0 males or F1 males and females), reduced weight gain in F0 males and females and F1 males (but not F1 females) and reduced food consumption in F0 males and females and F1 males (but not F1 females) during the ten-week pre-breed exposures was observed. With the exception of body weights and food consumption in F0 females, reductions in the pre-breed parameters appeared transitory, disappearing after one or two weeks. Reproductive parameters were not affected in either of the two breeds (F1 or F2). At initiation of the gestational period, body weights of the F0 (but not F1) females at 2000 pp, were reduced; weight gains through-out gestation for both breeds were normal. Reduced gestations food consumption was observed in F1 females only. F1 litters at 2000 ppm exhibited reduced body weights at weaning; both F1 and F2 generations of pups exhibited reduced body weights on Day 28 postpartum, one week subsequent to weaning. Body weight gains in both F1 and F2 litters were reduced for corresponding time intervals (lactational days 14-21 and 21-28) as well. The reduction in pup body weights (and weight gain) at 2000 ppm was considered to be treatment related as it corresponded to the time when the pups began to rely solely on the test substance diet as their source of nutrition. While the statistical reductions were observed only for female pup body weight gains on lactation days 21-28 in F1 litters and for male pups on days 14-21 in F2 litters, body weight gain reductions were apparent in both sexes of pups during these time periods. There were no treatment-related observations or histopathological findings in either the F0 or F1 adult animals at any dose. There were no treatment-related findings in the F1 or F2 pups that died during lactation or randomly selected F1and F2 pups at necropsy. Under the conditions of the study, it can be stated that, dietary administration of the test substance at dose levels of 0, 300, 1000 and 2000 ppm for two generations to Sprague-Dawley rats was well tolerated and no sign of reproductive toxicity was observed at any dose level. Also, there was no increased risk to the offspring in the absence of indications of maternal toxicity. The rat NOAEL (systemic toxicity) for both parental generation (P0 and F1) and offspring (F1 and F2) was considered to be at 1000 ppm (in diet), i.e., equivalent to 51-102 mg/kg bw/day (or 41-83 mg a.i./kg bw/day) in males and 67-106 mg/kg bw/day (or 54-86 mg a.i./kg bw/day) in females, respectively. The rat NOEL (reproductive toxicity) although not specified by the authors in the study report, can be considered to be at the highest tested dose 2000 ppm (in diet), i.e., equivalent to 100-188 mg/kg bw/day (or 81-152 mg a.i./kg bw/day) in males and 139-198 mg/kg bw/day (or 113-161 mg a.i./kg bw/day) in females (Neeper-Bradley, 1990).

Endpoint:
two-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2008
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to guideline
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Route of administration:
oral: feed
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
- Exposure period: about 18 wk.
P0 and P1:
Premating exposure period (males): 10 wk.
Premating exposure period (females): 10 wk.
- Duration of test: P0 pre-mating 10 wk, until F2 weaning.
Frequency of treatment:
Continuously
Details on study schedule:
Groups of 25 male and 25 female Sprague-Dawley rats were administered the test substance (purity 49.9%) at levels of 0, 500, 2000 or 4000 ppm in their diet over a period of 10 wk before mating, 2 wk during mating, and until after weaning of the pups (total period 18 wk). From every litter one or two pups were selected to again obtain 25 animals per sex and dose group for the F1 generation. These animals were similarly treated as the parent (P1) animals throughout premating, mating, pregnancy until sacrifice, after weaning of F2 progeny.
Remarks:
Doses / Concentrations: 0, 500, 2000 or 4000 ppm (equivalent to 250, 1000 and 2000 a.i. ppm) of test substance
Remarks:
F0 generation - 16-25, 61-101, 123-208 mg/kg bw/day (equivalent to 8-13, 31-51, 62-104 mg a.i./kg bw/day); F1 generation - 24-34, 96-123, 202-252 mg/kg bw/day (equivalent to 12-17, 48-62, 101-126 mg a.i./kg bw/day)
No. of animals per sex per dose:
25
Control animals:
yes, concurrent vehicle
Details on study design:
For details, kindly refer to the attached background material section of the IUCLID.
Parental animals: Observations and examinations:
- Examination of P0 and P1 generation:
Clinical signs and mortality were checked daily. Food consumption and body weight were recorded at designated intervals. Males and females were paired for a 2-wk period, until mating was obtained. The P1 females were allowed to deliver normally, and rear their progeny. Pregnancy and litter parameters were recorded.
- During lactation, the pups (F1 generation) were observed daily for survival and clinical signs; body weight was recorded at designated intervals; the sex-ratio was recorded. On Day 4 post-partum, the size of each litter was adjusted to obtain eight pups per litter (four males and four females).
- Reflex development was assessed at designated time-points.
Sperm parameters (parental animals):
Epididymal and testicular sperm parameters were evaluated for both P0 and P1 males.
Litter observations:
Examination of F1/P1 generation:
- On Day 22 post-partum, one male and one female pup per litter were selected to constitute the F1/P1 generation, which comprised 25 males and 25 females per group. The F1/P1 animals were observed daily for clinical signs and mortality. Body weight and food consumption were recorded once a week. Sexual development of both males and females was assessed.
- Neurobehavioural tests were conducted at designated intervals to assess auditory and visual functions. Spontaneous locomotor activity was also evaluated when the animals were between 7 and 8 wk old.
- After sexual maturity, F1 male and F1 female animals were paired. The F1 females were allowed to deliver normally, and rear their progeny. Pregnancy and litter parameters were recorded. During lactation, the pups (F2 generation) were observed daily for survival and clinical signs; body weight was recorded at designated intervals; the sex-ratio was recorded. On day 4 post-partum, the size of each litter was adjusted to obtain eight pups per litter (four males and four females).
- Reflex development was assessed at designated time-points.
Postmortem examinations (parental animals):
Terminal examination of P0 and P1 animals:
- After weaning of their respective progeny, P0 and P1 parent males and females were sacrificed. Designated organs were weighed for P0 and P1 parents, as well as brain, spleen and thymus of one pup per sex per litter of each generation.
- Epididymal and testicular sperm parameters were evaluated for both P0 and P1 males.
- A macroscopic post-mortem examination was performed on all P0 and P1 parent males and females. Any pups which died or were killed prematurely during the lactation period were also submitted for a macroscopic post-mortem examination. Macroscopic lesions, reproductive organs, adrenals and pituitary glands were sampled in all parent animals. A microscopic examination was performed on macroscopic lesions, reproductive organs, adrenals, and pituitary glands of all P0 and P1 parents of the control and high dose groups.
- Particularly detailed histopathological examinations were performed for the ovaries and the testes.
Postmortem examinations (offspring):
- A macroscopic post-mortem examination was performed on three pups per sex and per litter of each P0 and P1 (F1 and F2 generation) females killed at weaning. Any pups which died or were killed prematurely during the lactation period were also submitted for a macroscopic post-mortem examination. In all pups, the macroscopic lesions were preserved.
Statistics:
For details, kindly refer to the attached background material section of the IUCLID.
Reproductive indices:
For details, kindly refer to the attached background material section of the IUCLID.
Offspring viability indices:
For details, kindly refer to the attached background material section of the IUCLID.
Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- At 2000 ppm, a slightly lower mean body weight gain was recorded during most of the dosing period in both parental males and females (and was associated with reduced liver weights).
- At 1000 and 250 ppm, a marginally to slightly lower body weight gain was noted over all the dosing period for the males.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
- At 2000 ppm, a slightly to moderately lower mean food consumption was recorded during most of the dosing period in both parental males and females.
- At 1000 and 250 ppm, a marginally to slightly lower mean food consumption was noted over all the dosing period for the males.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
- At 2000 and 1000 ppm, no effects were noted at histopathological examination of sexual organs.

Histopathological findings: neoplastic:
not examined
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
- At 2000 and 1000 ppm, no effects were noted on sperm parameters.

Reproductive performance:
no effects observed
Description (incidence and severity):
- At 2000 ppm, slightly lower pup body weight was observed (with lower spleen weights).
- At 1000 ppm, no effects were observed on parental fertility as assessed by normal mating, gestation and delivery.
- At 250 ppm, no effects were noted on mating, fertility, gestation, fecundity or delivery of either generation or on development of their progeny.
As lower food consumption is known to occur due to palatability of compound. As no other substance related effects were seen than can be attributed to lower food intake, the level of 250 ppm test substance in the diet, corresponding to 16-25 and 24-31 mg/kg bw/day, should be regarded as NOAEL for P0 and P1generation respectively.
For further details, see under Any other information on results incl. tables.
Key result
Dose descriptor:
NOAEL
Remarks:
systemic toxicity
Effect level:
ca. 250 ppm
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
organ weights and organ / body weight ratios
gross pathology
Remarks on result:
other: equivalent to 16-25 mg/kg bw/day (8-12.5 mg a.i./kg bw/day)
Key result
Dose descriptor:
NOEL
Remarks:
reproductive toxicity
Effect level:
ca. 1 000 ppm
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: absence of adverse effects on mating behaviour, fertility and gestation of each generation
Remarks on result:
other: equivalent to 61-101 mg/kg bw/day (30.5-50.5 mg a.i./kg bw/day)
Key result
Critical effects observed:
no
Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- At 2000 ppm, a slightly to moderately lower mean body weight gain was recorded during most of the dosing period in both parental males and females (and was associated with reduced liver weights).
- At 1000 ppm, a marginally to slightly lower mean body weight gain was noted.
- At 250 ppm, a marginally to slightly lower mean body weight gain was observed in both sexes. This was associated with lower liver weights of parental males and females.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
- At 2000 ppm, a slightly to moderately lower mean food consumption was recorded during most of the dosing period in both parental males and females.
- At 1000 and 250 ppm, a marginally to slightly lower mean food consumption was noted over all the dosing period in both sexes.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
- At 2000 ppm, reduced liver weights were observed.
- At 1000 and 250 ppm, lower liver weights in parental animals were recorded.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
- At 2000 ppm, necropsy of these animals revealed dilation of the cecum, colon or ileum in some animals (more marked in F0 parents).
- At 1000 ppm, necropsy of parents revealed dilatation of the cecum in a single animal. 
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
- At 2000 and 1000 ppm, no effects were noted at histopathological examination of sexual organs.
Histopathological findings: neoplastic:
not examined
Details on results:
See under Any other information on results incl. tables for details on results.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
- At 2000 and 1000 ppm, no effects were noted on sperm parameters.

Reproductive performance:
no effects observed
Description (incidence and severity):
- At 2000 ppm, slightly lower pup body weight was observed for each progeny and was associated, for the F2 generation pups, with a reduction in litter size (as a consequence of lower number of implantation sites of P1 females) and a delay in sexual development. Lower spleen weights were also noted for each progeny.
- At 1000 ppm, no effects were noted on parental fertility as assessed by normal mating, gestation and delivery.
- At 250 ppm, no effects were noted on mating, fertility, gestation, fecundity or delivery and development of their progeny.
As lower food consumption is known to occur due to palatability of compound. As no other substance related effects were seen than can be attributed to lower food intake, the level of 250 ppm test substance in the diet, corresponding to 16-25 and 24-31 mg/kg bw/day, should be regarded as NOAEL for P0 and P1generation.
Key result
Dose descriptor:
NOAEL
Remarks:
systemic toxicity
Effect level:
ca. 250 ppm
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
organ weights and organ / body weight ratios
gross pathology
Remarks on result:
other: equivalent to 24-31 mg/kg bw/day (12-15.5 mg a.i./kg bw/day)
Key result
Dose descriptor:
NOEL
Remarks:
reproductive toxicity
Effect level:
ca. 1 000 ppm
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: absence of adverse effects on mating behaviour, fertility and gestation of each generation
Remarks on result:
other: corresponding to 96-123 mg/kg bw/day (48-61.5 mg a.i./kg bw/day)
Key result
Critical effects observed:
no
Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
- At 2000 ppm, the litter size at birth was reduced. 

Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- At 2000 ppm, slightly reduced pup body weight was observed. Pup weight gain was also slightly lower during lactation.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
- At 2000 ppm, lower spleen weights were noted.
- At 1000 ppm, except for a marginally lower spleen weight of the progeny, no other effects were noted on their development.
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
See under Any other information on results incl. tables for details on results.
Key result
Dose descriptor:
NOAEL
Remarks:
general toxicity
Generation:
F1
Effect level:
ca. 250 ppm
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: a marginally to slightly lower mean food consumption and body weight gain and liver weights
Remarks on result:
other: corresponding to 24-31 mg/kg bw/day (12-15.5 mg a.i./kg bw/day)
Key result
Dose descriptor:
NOAEL
Remarks:
developmental toxicity
Generation:
F1
Effect level:
ca. 1 000 ppm
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: mean age of preputial separation and vaginal opening was delayed due to reduced food intake
Remarks on result:
other: corresponding to 96-123 mg/kg bw/day (48-61.5 mg a.i./kg bw/day)
Key result
Critical effects observed:
no
Clinical signs:
not examined
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
- At 2000 ppm, the litter size at birth were reduced (as a consequence of lower number of implantation sites of F1 parent females).
- At 1000 ppm, no effects were seen in F2 offspring regarding pup survival until weaning.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- At 2000 ppm, slightly lower pup body weight was observed. The pup weight gain was slightly reduced during lactation, 
- At 1000 ppm, no effects were seen regarding pup development.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
- At 2000 ppm, lower spleen weights were noted.
- At 1000 ppm, except for a marginally lower spleen weight, no other effects were noted on their development.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
At 2000 ppm, dilatation of the caecum with faeces was observed in 4/25 males and 2/25 females.
At 1000 ppm, no effects were seen regarding pup development and after sacrifice at weaning.
Histopathological findings:
not examined
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Key result
Dose descriptor:
NOAEL
Remarks:
general / developmental toxicity
Generation:
F2
Effect level:
ca. 1 000 ppm
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
mortality
other: based on lower pup weights, lower pup weight gain during lactation, and upon necropsy dilatation of the cecum with feces was observed in 4/25 males and 2/25 females
Remarks on result:
other: corresponding to 96-123 mg/kg bw/day (48-61.5 mg a.i./kg bw/day)
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
no

The mean achieved dosages of the test substance for the dose-levels of 250, 1000 and 2000 ppm of test substance were as follows:
F0 generation
- males (Days 1 to 106): 16, 61 and 123 mg/kg bw/day, respectively,
- females:

during premating period (Days 1 to 71): 19, 74 and 154 mg/kg bw/day, respectively,
    during pregnancy period (Days 0 to 20 p.c.): 18, 69 and 145 mg/kg bw/day, respectively,
    during lactation period (Days 1 to 21 p.p.): 37, 159 and 326 mg/kg bw/day, respectively.
F1 generation
- males (Days 1 to 120): 24, 96 and 202 mg/kg bw/day, respectively,
- females:
    during premating period (Days 1 to 64): 32, 127 and 269 mg/kg bw/day, respectively,
    during pregnancy period (Days 0 to 20 p.c.): 21, 83 and 164 mg/kg bw/day, respectively,
    during lactation period (Days 1 to 21 p.p.): 41, 162 and 323 mg/kg bw/day, respectively.

The actual intake of test substance for both males and females given 250, 1000 and 2000 ppm throughout the study is approximately 16-25, 61-101 and 123-208 mg/kg bw/day, respectively  for the F0 generation and 24-31, 96-123 and 202-252 mg/kg bw/day for the F1 generation.

Details on results:

Parent males (F0):

No effects:mortality, clinical signs, mating and fertility indices, seminology parameters, macroscopic post-mortem examination and histopathology.

 

Body weight:

 

During the first week of dosing, the body weight gains were slightly low at the low and mid dose-levels (-13% and -11%, p<0.01, p<0.05, respectively) and moderately low at the high dose-level (days 1 to 8, -24%, p<0.001). This effect on body weight gain was observed over all the dosing period (days 1 to 106, - 11%, p<0.01; -8%, non-statistically significant and -20%, p<0.001, at 250, 1000 and 2000 ppm, respectively).

At all dose-levels, when compared to controls, body weights were marginally lower for the low and mid dose-levels (between -5 and -7%, p<0.01) and slightly lower for the high dose-level during most of the dosing period (-13%, p<0.001).

 

Food consumption:the food consumption during the premating period was marginally low at 250 and 1000 ppm reaching statistical significance during the first weeks of dosing (weeks 1, 3 and 4) and sporadically during 5 or 7 weeks (between -7% and -10%, p< 0.01, p<0.001). This effect was more marked at 2000 ppm and was statistically significant during the whole dosing period (between -7 and -14%, p<0.01, p<0.001).

 

Organ weights (expressed in %)

Lower absolute and body weight-related liver weights in the males given 1000 or 2000 ppm were statistically significant. In addition, the absolute value of the differences observed was of higher magnitude than that observed for the differences in body weights and the absolute and body weight-related differences were dose-related in the males. These differences were considered to be due to the treatment with the test substance, C12-16 ADBAC. It is worth mentioning that similar differences in liver weight without histomorphological changes were observed in the previous 90-day study.

 

Parent females F0

No effects:mortality, clinical signs, mating and fertility indices, estrous cycle, pregnancy and parturition data (implantation, fecundity, post-implantation, and neo-natal losses, gestation and delivery parameters) and histopathology.

Body weight:The body weight gain was unaffected by treatment at 250 and 1000 ppm. A moderately, statistically significantly, low body weight gain was observed at 2000 ppm during the first and the fourth week of premating (-31%, p<0.001 and -38%, p<0.01, respectively) and over all pregnancy (-13%, p<0.01) while it increased during the lactation period (24 g vs. 0 g, p<0.001). At this latter dose-level, body weights were lower during most of the dosing period (ranging between -4 and -9%, p<0.05, p<0.01) but returned to values comparable to controls at the end of the lactation period (316 g vs. 325 g in controls).

Food consumption:

The food consumption was unaffected by the treatment at 250 and 1000 ppm while it was slightly reduced at 2000 ppm, reaching statistical significance during the first week of dosing (-11%, p<0.001), during 2 weeks of the premating period (days 22 to 29 and days 64 to 71, -5% p<0.01) and during 2 weeks of the pregnancy period (days 0 to 7 p.c., -4%, p<0.05 and days 7 to 14,

-8%, p<0.001).

During the first week of the lactation period, group mean food consumption was slightly increased in the group given 2000 ppm and values were thereafter comparable to controls until the end of lactation.

 

Organ weights (expressed in %)

Lower absolute and body weight-related liver weights in females given 1000 or 2000 ppm were statistically significant. In addition, the absolute value of the differences observed was of higher magnitude than that observed for the differences in body weights. These differences were considered to be due to the treatment with the test item BKC. It is worth mentioning that similar differences in liver weight without histomorphological changes were observed in the previous 90-day study.

Macroscopic post-mortem examination

Some segments of the intestinal tract were distended with faeces at necropsy with higher incidence in the females given 2000 ppm, compared with their respective controls.

Although no microscopic finding could be correlated, considering the higher incidence of this finding in the test item-treated females compared with their respective controls, distension of intestinal tract was considered to be related to treatment with the test item. This included:

-         ileum in 1/25 females given 1000 ppm and 5/25 females given 2000 ppm vs. none control female,

-         coecum in 1/25 females given 250 ppm, 8/25 given 1000 ppm and 12/25 females given 2000 ppm vs. 2/25 control females,

-         colon in 1/25 females given 1000 ppm and 2/25 females given 2000 ppm vs. none control female.

F1 males

No effects:mortality, clinical signs, neurobehavioral tests, mating and fertility indices, seminology parameters and histopathology.

Body weight:On day 1 (post-natal day 22), the body weight was slightly lower at 2000 ppm (-13%, p<0.001) while it was minimally, but not statistically significantly,

affected at 1000 ppm (-5%) and was similar to controls at 250 ppm. During the whole dosing period, the body weight was generally slightly (for the low and mid-dose levels) to moderately (for the high dose-level), but statistically significantly, lower. The body weight gain was statistically significantly lower during the two first weeks of dosing at all dose-levels and remained markedly lower over all the dosing period at the high dose-level (days 1 to 120, - 18%,p<0.001).

Food consumption:

In the males, group mean food consumption was slightly to moderately lower at all dose-levels (ranging between -7 and -21%, p<0.05, p<0.01, p<0.001) reaching statistical significance during many periods at the low and mid dose- levels and during all recorded periods at the high dose-level (p<0.01, p<0.001).

Sexual development

The mean age at preputial separation (cleavage of the balanopreputial gland) was unaffected by the treatment at 250 and 1000 ppm (i.e occurred at 49.6 and

50.1 day old, respectively vs. 47.8 day old in controls) while, when compared to the male control group, a significant difference was observed in males given 2000 ppm (52.80 ± 6.02 vs. 47.76 ± 2.62 days, p<0.0019). It was not statistically significant when body weight was considered as a covariate, which suggests that this effect was the consequence of a lower body weight rather than a direct effect on sexual development.

Organ weights (expressed in %)

Lower absolute and/or body weight-related liver weights in the males given 250, 1000 or 2000 ppm were observed, with statistical significance. In addition, the absolute value of the differences observed was of higher magnitude than that observed for the differences in body weights. These differences were therefore considered to be due to treatment with the test item, BKC. It is worth mentioning that similar differences in liver weight without histomorphological changes were observed in the previous 90-day study and similar differences in liver weights were also noted in the F0 generation.

Macroscopic post-mortem examination

The coecum was distended with faeces in 1/25 males given 1000 ppm and in 4/25 males given 2000 ppm.

Although no microscopic finding could be correlated (at least for the macroscopic abnormalities which were examined), considering the previous observations made in the intestinal tract in the females of the F0 generation, this finding reported in a few test item-treated animals of both sexes, was considered to be most probably related to treatment with the test item.

 

F1 females

No effects:mortality, clinical signs, neurobehavioral tests, mating and fertility indices, oestrous cycle, pregnancy and parturition data (with the exception of number of implantation sites and number of live foetuses), and histopathology.

Body weight:On day 1 (post-natal day 22), the body weight was statistically significantly lower at 1000 and 2000 ppm (-6%, p<0.005 and -13%, p<0.001, respectively) and remained lower over all the premating and pregnancy periods until the second week of lactation. The body weight gain was marginally reduced (-9%, not statistically significant and -15% p<0.001, for the low and mid-dose-levels, respectively) to markedly reduced (-41%, p<0.001 for the high dose-level) during the first week of premating and over the pregnancy periods (-10%, -11%, -19%, respectively, p<0.01 or p<0.001) while it was increased when compared to controls during the lactation period.

Food consumption:

In the females, group mean food consumption was unaffected by the treatment at the low and mid dose-groups while it was lower at 2000 ppm during premating and pregnancy, reaching statistical significance during most of the recorded periods (ranging between -9 and -14%, p<0.01, p<0.001). No effect was noted during the lactation period.

Sexual development

The mean age for vaginal opening was unaffected by the treatment, i.e. occurred on days 34.1, 33.7, 35.4 for pups from the control, 250 and 1000 ppm groups, respectively while it was delayed for 4 days at 2000 ppm.

When compared to the female control group, a significant difference was observed in females given 2000 ppm (38.40 ± 5.16 vs. 34.12 ± 2.19 in controls p<0.0001), however it was not statistically significant when body weight was considered as a covariate which suggests that this effect was the consequence of a lower body weight rather than a direct effect on sexual development.

Furthermore, animals that had values outside the range of individual control values had the lowest weight at sexual maturity which confirm that this effect was the consequence of reduced body weight and not a direct effect on sexual development.

Pregnancy and parturition data

The statistically significantly lower number of implantation sites and the lower number of live foetuses (-13%, p<0.001 for both parameters) in females given 2000 ppm when compared to controls, were considered to be treatment- related, although these values were within the range of historical control data.

Organ weights (expressed in %)

Lower absolute and/or body weight-related liver weights in the females given 250, 1000 or 2000 ppm were observed, with statistical significance and dose- relationship in the females. In addition, the absolute value of the differences observed was of higher magnitude than that observed for the differences in body weights. These differences were therefore considered to be due to treatment with the test item, BKC. It is worth mentioning that similar differences in liver weight without histomorphological changes were observed in the previous 90-day study and similar differences in liver weights were also noted in the F0 generation.

Macroscopic post-mortem examination

The cecum was distended with feces in 2/25 females given 2000 ppm. Although no microscopic finding could be correlated (at least for the macroscopic abnormalities which were examined), considering the previous observations made in the intestinal tract in the females of the F0 generation, this finding reported in a few test item-treated animals of both sexes, was considered to be most probably related to treatment with the test item.

 

F2 males

No effects:viability and lactation indices, clinical signs, gross external abnormalities, assessment of reflex development (surface righting, cliff avoidance and air righting) and macroscopic post-mortem examination.

Body weight:No effects on body weight or body weight gain were noted at 250 and 1000 ppm while slightly lower body weight gain was observed at 2000 ppm between day 4 and day 21 post-partum (-8%, p<0.05).

Although this difference in body weight gain was slight, it was considered to be possibly related to the treatment.

Organ weights (expressed in %)

Considering that the differences in absolute and body weight-related weights of the spleen of the animals given 1000 or 2000 ppm were dose-related in the two sexes and that the absolute value of the difference in the absolute spleen weight was marginally higher than that observed for the difference in body weight at necropsy, a relationship to treatment could not be excluded, as similar changes were observed in the spleen weights of the pups of the F1 generation.

Macroscopic post-mortem examination

The cecum was distended with feces in 2/25 females given 2000 ppm. Although no microscopic finding could be correlated (at least for the macroscopic abnormalities which were examined), considering the previous observations made in the intestinal tract in the females of the F0 generation, this finding reported in a few test item-treated animals of both sexes, was considered to be most probably related to treatment with the test item.

F2 females

No effects:viability and lactation indices, clinical signs, gross external abnormalities, assessment of reflex and physical development (surface righting, cliff avoidance and air righting) and macroscopic post-mortem examination.

Body weight:No effects on body weight or body weight gain were noted at 250 and 1000 ppm while slightly lower body weight gain was observed at 2000 ppm between day 4 and day 21 post-partum (-8%, p<0.05).

Although this difference in body weight gain was slight, it was considered to be possibly related to the treatment.

Organ weights (expressed in %)

Considering that the differences in absolute and body weight-related weights of the spleen of the animals given 1000 or 2000 ppm were dose-related in the two sexes and that the absolute value of the difference in the absolute spleen weight was marginally higher than that observed for the difference in body weight at necropsy, a relationship to treatment could not be excluded, as similar changes were observed in the spleen weights of the pups of the F1 generation.

Offspring of F0 parents (F1 pups)

No effects:viability and lactation indices, clinical signs and gross external abnormalities, assessment of reflex development and macroscopic post-mortem examination.

Body weight:At birth, pup body weights were unaffected by the treatment at all dose-levels. During the lactation period, pup body weight and body weight gain were not affected at 250 ppm.

At 1000 and 2000 ppm, pup body weight gains were statistically significantly lower during the last week of lactation (-9%, p<0.05 and -11%, p<0.01) while body weight was statistically significantly lower on day 21 post-partum for the high-dose only (-7%, p<0.05). This effect was considered to be most probably related to pup food intake (which normally started during the last week of lactation) and was therefore considered to be related to the treatment.

Organ weights (pups sacrificed at weaning)

Dose-related lower absolute and body weight-related spleen weights were observed in the male and female pups given 1000 or 2000 ppm. This achieved statistical significance for the absolute weight for the animals given 2000 ppm. Almost all the individual weights were within the control range for the test item-treated animals; however they were gathered at the lower limit of the range, especially at the high dose-level.

For the differences in spleen weights, a relationship to treatment with the test item cannot be ruled out, as the same trend was observed in the pups of the F1 generation. However, they might be stress-related, considering the young age of the animals and/or the consequence, at least in part, of the decrease in body weight.

 

Results for F0 and F1 generation summarised according to dose groups:

The following results were obtained:

F0 generation (during premating, mating, pregnancy and lactation until day 21 post-partum of F0 parents and post-natal day 21 for their progeny)

Group 4: 2000 ppm

-        no treatment related clinical signs or mortality were noted,

-        for the parent males, over all the dosing period, statistically significantly low food consumption (between -7 and -14%, p<0.01 and p<0.001), low body weights (-6 and -13%, p<0.001) and reduced body weight gains (- 20%, p<0.001) were noted,

-        for the parent females, the food consumption was reduced during the first week of the premating period (-11%, p<0.001) and during the 2 first weeks of pregnancy (-9% and -8%, respectively). This was associated with lower body weight gain during the first week of the premating period (-31%, p<0.001), during the pregnancy period (-13%, p<0.01) and lower body weights over all the premating and pregnancy periods (between -4% and -9%, p<0.05, p<0.01). The food consumption and the body weight gains were slightly increased during the first week of lactation,

-        no effect on mating, male and female fertility (including estrous cycle and seminology parameters) or on the progress of pregnancy or delivery,

-        no effect on pup survival (F1 generation pups) while reduced pup body weight gain was noted during the last week of lactation (-11%, p<0.01),

-        necropsy of parents revealed dilatation of ileum in 5/25 females, dilatation of cecum in 12/25 females and of the colon in 2/25 females,

-        lower absolute and body weight-related liver weights in parental males (respectively, -23%, -11%, p<0.01) and females (respectively, -16 % and

-        11%, p<0.01) and lower absolute and relative spleen weights of their progeny (respectively, -16%, p<0.05 and -9% for the males, -14%, p<0.05 and -8% for the females),

-        no histopathological findings were noted at microscopic examination of  the different organs (including reproductive organs).

 

Group 2 and 3: 250 and 1000 ppm

-        no treatment related deaths or clinical signs were observed,

-        for the parent males, the food consumption was marginally low during the first week of dosing and sporadically during 4 or 5 weeks (between -7% and -10%, p<0.01, p<0.001) and was associated with a slightly low body weight gain during the first week of dosing (-13% and -11%, p<0.01, p<0.05, respectively) and over all the dosing period (-11%, p<0.05; -8%, respectively at both dose-levels),

-        for the parent females, the food consumption and body weight gains were unaffected by the treatment,

-        no effect on mating, male and female fertility (including estrous cycle and seminology parameters), gestation, fecundity or delivery,

-        no effect on progeny survival from delivery until weaning (F1 generation pups),

-        necropsy of parental males and females given 1000 ppm revealed dilatation of the cecum with feces in 8/25 females and dilatation of the ileum and the colon in 1/25 females,

-        at 1000 ppm, there were lower absolute and body weight-related liver weights of parental males (respectively, -12%, p<0.05 and -7%, p<0.05) and females (-14% and -11%, p<0.01) and marginal, non-statistically significant, lower absolute and body weight-related progeny spleen weights (-7% and -4% for the males and -9% and -6% for the females),

-        at 250 ppm, no effect was noted on parents or progeny‟ organ weights,

-        no histopathological findings were noted at microscopic examination of  the different parental organs (including reproductive organs) at either dose-level.

 

F1 generation (from post-natal day 22, during mating, pregnancy and lactation until day 21 post-partum)

Group 4: 2000 ppm

-        no treatment-related clinical signs or mortality in parental males or females,

-        for the males, the food consumption was lower during the whole dosing period (between -12 and -21%, p<0.0001) and the body weight and body weight gain were moderately lower over all the dosing period (-18%, p<0.001),

-        for the females, reduced food consumption was noted during the premating and pregnancy periods (ranging between -9% and -14%, p<0.01 and p<0.001). This was associated with lower body weight from the first day of the premating period until the second week of lactation and lower body weight gain during the first week of premating (-41%, p<0.001) and over all the premating and pregnancyperiods,

-        the mean age of preputial separation and vaginal opening was delayed   for approximately 5 and 4 days, respectively (52.8 vs. 47.76 day old for preputial separation and 38.4 vs. 34.12 day old for vaginal opening) as a consequence of lower body weight, while no effect was noted on their locomotor activity or on neurobehavioralparameters,

-        no effects were noted on mating or parental fertility (including number of pregnant females with liveborn, estrous cycle and seminology parameters), however, the number of implantation sites and the litter size at birth were reduced (13 vs. 14.9 and 12.4 vs. 14.2, p<0.01, respectively),

-        the pup weight gain of the progeny of the F1 generation (F2 generation pups) was slightly lower during the lactation period (-8%,p<0.05),

-        necropsy of the F1 generation revealed dilatation of the cecum with feces in 4/25 males and 2/25females,

-        lower absolute and body weight-related liver weights of parental males (respectively, -28% and -12%, p<0.01) and females (respectively, -25% and -18%, p<0.01) and decreased absolute and relative spleen weights of their progeny (-23%, p<0.01 and -10% for the males, -26% and -14%, p<0.01 for the females),

-        no histopathological findings were noted at microscopic examination of  the different organs (including reproductiveorgans).

 

Group 3: 1000 ppm

-        no treatment-related clinical signs in parental males andfemales,

-        for the males, the food consumption was statistically significantly lower during many dosing periods (between -6% and -11%, p<0.05, p<0.01, p<0.001) and was associated with a slightly lower body weight and body weight gain over all the dosing period (-7%),

-        for the females, the body weight and food consumption were slightly lower during the first week of premating and during pregnancy (body weight: -15%, p<0.001 and -11%, p<0.01, food consumption: between -9 and -14%, p<0.01,p<0.001),

-        no effect on mating, parental fertility (including estrous cycle and seminology parameters), gestation, fecundity ordelivery,

-        no effect on pup development and progeny survival from delivery until weaning,

-        no effect on locomotor activity and neurobehavioural parameters of the progeny,

-        necropsy of the parents revealed dilatation of coecum with faeces in 1/25 males,

-        lower absolute and body weight-related liver weights of parental males (respectively, -13%, p<0.05 and -6%, not statistically significant) and females (respectively, -17% and -14%, p<0.01) and marginally lower absolute and body weight-related spleen weight of their progeny (-3% for the males and -11 and -10%, respectively for the females),

-        no histopathological findings were noted at microscopic examination of  the different organs (including reproductive organs).

Group 2: 250 ppm

-        no effects at clinical examination of the male and female parents,

-        for the males, body weight and body weight gain were slightly, but statistically significantly, lower during most of the dosing period (-9%, p<0.05) while the food consumption was transiently lower,

-        for the females, the body weight gain was slightly lower during pregnancy (days 0 to 20 p.c., -10%, p<0.01) while no effect was noted on food consumption,

-        no effect on mating, male and female parental fertility (including estrous cycle and seminology parameters), gestation, fecundity or delivery,

-        no effect on sexual development or on neurobehavioral parameters,

-        no effect on the development of the progeny from delivery until weaning,

-        no macroscopic findings in parental males and females or their progeny,

-        lower absolute and body weight-related liver weights of parental males (respectively, -14%, p<0.05 and -6%, not statistically significant) and females (respectively, -14% and -12%, p<0.01) but no effect was observed on organ weights of the progeny,

-        no microscopic findings for male and female parents.

Conclusions:
Under the study conditions, the rat NOAEL (systemic toxicity) for both parental generation (F0 and F1) was considered to be at 250 ppm [equivalent to 16-25 mg/kg bw/day (or 8-12.5 mg a.i./kg bw/day) for F0 and 24-31 mg/kg bw/day (or 12-15.5 mg a.i./kg bw/day) for F1 generation, respectively]. The rat NOEL (reproductive toxicity) and NOAEL (developmental toxicity) was considered to be at 1000 ppm [equivalent to 61-101 mg/kg bw/day (or 30.5-50.5 mg a.i./kg bw/day) for F0 and 96-123 mg/kg bw/day (or 48-61.5 mg a.i./kg bw/day) for F1 generation, respectively] (Foulon, 2008).
Executive summary:

A two-generation study was conducted to determine the toxicity to reproduction of the test substance, C12 -16 ADBAC (49.9% active in water) according to OECD Guideline 416, in compliance with GLP. In this study, the substance was administered in the diet to male and female Sprague-Dawley rats at dose levels of 0, 500, 1000 and 4000 ppm (corresponding to 0, 250, 1000 and 2000 ppm a.i. and equivalent to 0, 16-25, 61-101 and 123- 208 mg a.i./kg bw/day, respectively for the F0 generation and 24-31, 96-123 and 202-252 mg a.i./kg bw/day for the F1 generation respectively). Doses were administered before and throughout mating and gestation until the end of the lactation period in both F0 and F1 generations. At 2000 ppm, a slightly to moderately lower mean food consumption and mean body weight gain were recorded during most of the dosing period in both parental males and females of the two generations and was associated with reduced liver weights. Necropsy of these animals (parents of both generations) revealed dilatation of the cecum, colon or ileum in some animals (more marked in F0 parents). No effects were noted on sperm parameters or on histopathological examination of sexual organs. Slightly lower pup body weight was observed for each progeny and was associated, for the F2 generation pups, with a reduction in litter size (as a consequence of lower number of implantation sites of F1 parent females) and a delay in sexual development. Lower spleen weights were also noted for each progeny. At 1000 ppm, P0 (males) and P1 (both sexes) showed a marginally to slightly lower mean food consumption and body weight gain were noted over all the dosing period for the males of the F0 generation and in both sexes for the F1 generation. Necropsy of parents of both generations revealed dilatation of the cecum in some animals of the F0 generation and in a single animal of the F1 generation. This was associated with lower liver weights in parental animals of both generations. No effects were noted on parental fertility as assessed by normal mating, gestation and delivery and, particularly, there were no effects on sperm parameters or at histopathological examination of sexual organs. Except for a marginally lower spleen weight of the progeny of each generation, no other effects were noted on their development. At 250 ppm (corresponding to approximately 16-25 mg a.i./kg bw/day for F0 males and females and 24-31 mg a.i./kg bw/day for F1 males and females, respectively), a marginally to slightly lower mean food consumption and body weight gain were noted over all the dosing period for the males of the F0 generation and in both sexes for the F1 generation. This was associated in the F1 generation with lower liver weights of parental males and females. No effects were noted on mating, fertility, gestation, fecundity or delivery of either generation or on development of their progeny.The lower food intake, which was most pronounced in period of start of intake as well in F0 as in F1 animals, indicated that the intake was influenced by palatability of the compound and not by a toxic mechanism. At 250 ppm, the small effects on body weight, body weight changes and absolute and relative organ weights were considered to be resulting from reduced food intake. Similar was the case for the 1000 ppm group. As this was not considered to be an adverse toxic effect from exposure to the test substance, 250 ppm was considered to be the NOAEL for parental systemic toxicity. The NOEL for mating behaviour, fertility and gestation of each generation was considered to be 1000 ppm. The marginally lower absolute and body weight-related spleen weight of the F2 progeny at 1000 ppm was also considered to be indirectly caused by the lower food intake of F1 parents, and therefore was not considered to be an adverse toxic effect from exposure to the test substance. Therefore, the NOAEL for development, growth and survival of each progeny was considered to be at 1000 ppm. Under the study conditions, the rat NOAEL (systemic toxicity) for both parental generation (F0 and F1) was considered to be at 250 ppm [equivalent to 16-25 mg/kg bw/day (or 8-12.5 mg a.i./kg bw/day) for F0 and 24-31 mg/kg bw/day (or 12-15.5 mg a.i./kg bw/day) for F1 generation, respectively]. The rat NOEL (reproductive toxicity) and NOAEL (developmental toxicity) was considered to be at 1000 ppm [equivalent to 61-101 mg/kg bw/day (or 30.5-50.5 mg a.i./kg bw/day) for F0 and 96-123 mg/kg bw/day (or 48-61.5 mg a.i./kg bw/day) for F1 generation, respectively] (Foulon, 2008).

Endpoint:
two-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1989
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
secondary literature
Principles of method if other than guideline:
Guideline not mentioned.
GLP compliance:
no
Species:
other: rats and guinea pigs
Sex:
male/female
Route of administration:
oral: gavage
Analytical verification of doses or concentrations:
not specified
Dose / conc.:
25 mg/kg bw/day
No. of animals per sex per dose:
15 rats and 10 guinea pigs
Key result
Reproductive effects observed:
no

A 2 generation study with groups of 15 rats and 10 guinea pigs was conducted with up to 25 mg/kg bw/day by gavage and showed no overt adverse effects on reproduction.

Conclusions:
Under the study conditions, no adverse effects on reproduction were reported in a 2-generation study conducted in groups of 15 rats and 10 guinea pigs, up to the dose 25 mg/kg bw/day (oral gavage).
Executive summary:

A two-generation study was conducted in groups of 15 rats and 10 guinea pigs up to the dose of 25 mg/kg bw/day test substance (administered by stomach tube). Under the study conditions, no overt adverse effects on reproduction were observed (Shelanski, 1989).

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
16 mg/kg bw/day
Study duration:
subchronic
Experimental exposure time per week (hours/week):
168
Species:
rat
Quality of whole database:
The information requirement for this tonnage band is sufficiently met with the available data.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

Study 1:A two-generation study was conducted to determine the toxicity to reproduction of the test substance, C12 -16 ADBAC (49.9% active in water) according to OECD Guideline 416, in compliance with GLP. In this study, the substance was administered in the diet to male and female Sprague-Dawley rats at dose levels of 0, 500, 1000 and 4000 ppm (corresponding to 0, 250, 1000 and 2000 ppm a.i. and equivalent to 0, 16-25, 61-101 and 123- 208 mg a.i./kg bw/day, respectively for the F0 generation and 24-31, 96-123 and 202-252 mg a.i./kg bw/day for the F1 generation respectively). Doses were administered before and throughout mating and gestation until the end of the lactation period in both F0 and F1 generations. At 2000 ppm, a slightly to moderately lower mean food consumption and mean body weight gain were recorded during most of the dosing period in both parental males and females of the two generations and was associated with reduced liver weights. Necropsy of these animals (parents of both generations) revealed dilatation of the cecum, colon or ileum in some animals (more marked in F0 parents). No effects were noted on sperm parameters or on histopathological examination of sexual organs. Slightly lower pup body weight was observed for each progeny and was associated, for the F2 generation pups, with a reduction in litter size (as a consequence of lower number of implantation sites of F1 parent females) and a delay in sexual development. Lower spleen weights were also noted for each progeny. At 1000 ppm, P0 (males) and P1 (both sexes) showed a marginally to slightly lower mean food consumption and body weight gain were noted over all the dosing period for the males of the F0 generation and in both sexes for the F1 generation. Necropsy of parents of both generations revealed dilatation of the cecum in some animals of the F0 generation and in a single animal of the F1 generation. This was associated with lower liver weights in parental animals of both generations. No effects were noted on parental fertility as assessed by normal mating, gestation and delivery and, particularly, there were no effects on sperm parameters or at histopathological examination of sexual organs. Except for a marginally lower spleen weight of the progeny of each generation, no other effects were noted on their development. At 250 ppm (corresponding to approximately 16-25 mg a.i./kg bw/day for F0 males and females and 24-31 mg a.i./kg bw/day for F1 males and females, respectively), a marginally to slightly lower mean food consumption and body weight gain were noted over all the dosing period for the males of the F0 generation and in both sexes for the F1 generation. This was associated in the F1 generation with lower liver weights of parental males and females. No effects were noted on mating, fertility, gestation, fecundity or delivery of either generation or on development of their progeny. The lower food intake, which was most pronounced in period of start of intake as well in F0 as in F1 animals, indicated that the intake was influenced by palatability of the compound and not by a toxic mechanism. At 250 ppm, the small effects on body weight, body weight changes and absolute and relative organ weights were considered to be resulting from reduced food intake. Similar was the case for the 1000 ppm group. As this was not considered to be an adverse toxic effect from exposure to the test substance, 250 ppm was considered to be the NOAEL for parental systemic toxicity. The NOEL for mating behaviour, fertility and gestation of each generation was considered to be 1000 ppm. The marginally lower absolute and body weight-related spleen weight of the F2 progeny at 1000 ppm was also considered to be indirectly caused by the lower food intake of F1 parents, and therefore was not considered to be an adverse toxic effect from exposure to the test substance. Therefore, the NOAEL for development, growth and survival of each progeny was considered to be at 1000 ppm. Under the study conditions, the rat NOAEL (systemic toxicity) for both parental generation (F0 and F1) was considered to be at 250 ppm [equivalent to 16-25 mg/kg bw/day (or 8-12.5 mg a.i./kg bw/day) for F0 and 24-31 mg/kg bw/day (or 12-15.5 mg a.i./kg bw/day) for F1 generation, respectively]. The rat NOEL (reproductive toxicity) and NOAEL (developmental toxicity) was considered to be at 1000 ppm [equivalent to 61-101 mg/kg bw/day (or 30.5-50.5 mg a.i./kg bw/day) for F0 and 96-123 mg/kg bw/day (or 48-61.5 mg a.i./kg bw/day) for F1 generation, respectively] (Foulon, 2008).

 

Study 2:A two-generation study was conducted to determine the toxicity to reproduction of the test substance, C12-16 ADBAC (81.09% active in aqueous/ethanol solution) according to US EPA OPP 83-4, in compliance with GLP. The test substance was orally administered to Sprague-Dawley CD rats (28/sex/group) at dose levels of 0, 300, 1000 or 2000 ppm test substance (equivalent to average doses of 0, 16-31, 51-102, and 100-188 mg/kg bw/day for males and 0, 21-32, 67-106 and 139-198 mg/kg bw/day in females) in the diet. Following a 10-week pre-breed exposure period, the P0 rats were randomly paired within dose groups for a 3-week mating period to produce the F1 generation. Exposure continued through mating, gestation, parturition and lactation. At weaning, twenty-eight (28) F1 weanling/sex/group were randomly selected and exposed to the same dietary concentration of the test substance as their parents for 10 weeks. After their pre-breed exposure, F1/P1 animals were paired to produce the F2 generation. The animals were observed twice daily and clinical findings, body weights and food consumption were recorded periodically. All P0 and F1 animals were necropsied and examined for gross lesions. Selected reproductive tissues from the high dose and control groups were examined histologically as were other tissues with gross lesions. Ten F1 and F2 weanlings/sex/dose were randomly selected and necropsied and examined for gross lesions. Remaining non-selected F1 and F2 pups at weaning were euthanised and discarded. Reduced body weights in F0 males (but no F0 males or F1 males and females), reduced weight gain in F0 males and females and F1 males (but not F1 females) and reduced food consumption in F0 males and females and F1 males (but not F1 females) during the ten-week pre-breed exposures was observed. With the exception of body weights and food consumption in F0 females, reductions in the pre-breed parameters appeared transitory, disappearing after one or two weeks. Reproductive parameters were not affected in either of the two breeds (F1 or F2). At initiation of the gestational period, body weights of the F0 (but not F1) females at 2000 pp, were reduced; weight gains through-out gestation for both breeds were normal. Reduced gestations food consumption was observed in F1 females only. F1 litters at 2000 ppm exhibited reduced body weights at weaning; both F1 and F2 generations of pups exhibited reduced body weights on Day 28 postpartum, one week subsequent to weaning. Body weight gains in both F1 and F2 litters were reduced for corresponding time intervals (lactational days 14-21 and 21-28) as well. The reduction in pup body weights (and weight gain) at 2000 ppm was considered to be treatment related as it corresponded to the time when the pups began to rely solely on the test substance diet as their source of nutrition. While the statistical reductions were observed only for female pup body weight gains on lactation days 21-28 in F1 litters and for male pups on days 14-21 in F2 litters, body weight gain reductions were apparent in both sexes of pups during these time periods. There were no treatment-related observations or histopathological findings in either the F0 or F1 adult animals at any dose. There were no treatment-related findings in the F1 or F2 pups that died during lactation or randomly selected F1and F2 pups at necropsy. Under the conditions of the study, it can be stated that, dietary administration of the test substance at dose levels of 0, 300, 1000 and 2000 ppm for two generations to Sprague-Dawley rats was well tolerated and no sign of reproductive toxicity was observed at any dose level. Also, there was no increased risk to the offspring in the absence of indications of maternal toxicity. The rat NOAEL (systemic toxicity) for both parental generation (P0 and F1) and offspring (F1 and F2) was considered to be at 1000 ppm (in diet), i.e., equivalent to 51-102 mg/kg bw/day (or 41-83 mg a.i./kg bw/day) in males and 67-106 mg/kg bw/day (or 54-86 mg a.i./kg bw/day) in females, respectively. The rat NOEL (reproductive toxicity) although not specified by the authors in the study report, can be considered to be at the highest tested dose 2000 ppm (in diet), i.e., equivalent to 100-188 mg/kg bw/day (or 81-152 mg a.i./kg bw/day) in males and 139-198 mg/kg bw/day (or 113-161 mg a.i./kg bw/day) in females (Neeper-Bradley, 1990).

Study 3:A two-generation study was conducted in groups of 15 rats and 10 guinea pigs up to the dose of 25 mg/kg bw/day test substance (administered by stomach tube). Under the study conditions, no overt adverse effects on reproduction were observed (Shelanski, 1989).

The biocides assessment reports available from RMS Italy on C12-16 ADBAC and Coco TMAC, overall concluded that these substances do not indicate a concern for reproduction toxicity. However, the corrected dietary doses were slightly different in the C12-16 ADBAC biocides assessment report, where the NOAELs for parental and offspring toxicity were both reported be >50 and >30 mg/kg bw/day and the NOAELs for effects on fertility was reported to be >100 and >52 mg/kg bw/day for the Foulon, 2008 and Neeper-Bradley, 1990 studies respectively (ECHA biocides assessment report, 2015, 2016). The assessment report on Coco TMAC, which contained only the Foulon 2008 study however corresponded to the NOAELs summarized in this dossier. Nevertheless, due to the absence of specific reproductive toxicity and the fact that the observed effects in parents relate to general toxicity which are secondary to local effects, the parental NOAELs (16 mg/kg bw/day) were not considered to be relevant to the determination of systemic DNELs.

 

In line with the biocides assessment report, the NOAELs from the 2-generation reproductive toxicity studies in rats have not been considered further for systemic risk assessment.

Effects on developmental toxicity

Description of key information

The available oral and dermal pre-natal development toxicity study with the test substances in rats and rabbits, indicate no concern for developmental toxicity.

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Remarks:
Dose range finding study
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2004
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The study was performed according to the range finding study design of OECD guideline 414.
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
Dose range finding study
Deviations:
no
GLP compliance:
no
Specific details on test material used for the study:
Test material details of this DRF study is same as the Gaoua 2005 study
Species:
rabbit
Strain:
New Zealand White
Route of administration:
oral: gavage
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
Day 6 to 28 post coitum
Frequency of treatment:
Daily
Dose / conc.:
0 mg/kg bw/day
Dose / conc.:
3 mg/kg bw/day
Dose / conc.:
10 mg/kg bw/day
Dose / conc.:
30 mg/kg bw/day
No. of animals per sex per dose:
6 females per dose
Control animals:
yes, concurrent vehicle
Details on study design:
Sex: male/female
Details on maternal toxic effects:
At 30 mg/kg bw/day, slightly higher mean number of post-implantation loss associated with slightly lower mean number of live foetuses were recorded. No maternal toxicity or effects on litter data parameters were noted at 3 and 10 mg/kg bw/day.
Key result
Dose descriptor:
NOAEL
Remarks:
systemic toxicity
Effect level:
10 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: no general maternal toxicity was observed at 3 and 10 mg/kg bw/day
Key result
Dose descriptor:
NOAEL
Remarks:
reproductive toxicity
Effect level:
10 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
dead fetuses
pre and post implantation loss
Key result
Abnormalities:
effects observed, treatment-related
Localisation:
other: post-implantation sites and foetuses viability
Key result
Dose descriptor:
NOAEL
Remarks:
general and developmental toxicity
Effect level:
10 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reduction in number of live offspring
Key result
Abnormalities:
effects observed, treatment-related
Localisation:
other: foetuses viability
Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
30 mg/kg bw/day
Treatment related:
yes
Relation to maternal toxicity:
developmental effects as a secondary non-specific consequence of maternal toxicity effects
Dose response relationship:
yes

At 30 mg/kg bw/day, slightly high mean number of post-implantation loss associated with slightly low mean number of live foetuses were recorded. No maternal toxicity or effects on litter data parameters were noted at 3 and 10 mg/kg bw/day.

Based on these results, the 30 mg/kg bw/day dose-level was chosen as the high dose-level in the further developmental toxicity study (CIT Study No. 26148 RSL).

Conclusions:
Under the study conditions (dose range finding study), a slightly higher mean number of post-implantation loss associated with slightly lower mean number of live foetuses was recorded at 30 mg/kg bw/day. No maternal toxicity or effects on litter data parameters were noted at 3 and 10 mg a.i./kg bw/day. Based on these results, the 30 mg/kg bw/d dose-level was chosen as the high dose-level in the further developmental toxicity study.
Executive summary:

A dose range-finding study was conducted to determine the doses needed for conducting developmental toxicity and teratogenicity stud with the test substance (49.9% active in water) according to OECD Guideline 414 (see below). Groups of six mated female rabbits received daily oral administration at 0 (vehicle, purified water), 3, 10 or 30 mg/kg bw/day from Day 6 to 28 post-coitum. In the total absence of maternal toxicity at all dose levels and to better define the top dose level of the main study, the study director suggested to perform a complementary study with higher dose levels of 50 and 100 mg/kg bw/day. The test substance from GD 6-28 produced marked maternal toxicity at 50 and 100 mg/kg bw/day shown by high number of deceased females and severe clinical conditions in most animals. At 30 mg/kg bw/day, a slightly higher mean number of post-implantation loss associated with a slightly lower mean number of live foetuses was recorded. No maternal toxicity or effects on litter data parameters were noted at 3 and 10 mg/kg bw/d. Based on these results, the 30 mg/kg bw/day dose level was chosen as the high dose level in the further developmental toxicity study (Gaoua, 2004).

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
batch number is not presented for the vehicle (purified water) in the study report; autolysed foetuses were not sexed at the time of hysterectomy and head sections were archived in the same way as brain sections.
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
batch number is not presented for the vehicle (purified water) in the study report; autolysed foetuses were not sexed at the time of hysterectomy and head sections were archived in the same way as brain sections.
GLP compliance:
yes
Limit test:
no
Species:
rabbit
Strain:
New Zealand White
Remarks:
KBL New Zealand White, Specific Pathogen Free (S.P.F.).
Details on test animals or test system and environmental conditions:
- Source: Charles River Laboratories (Elevage Scientifique des Dombes, Châtillon-sur- Chalaronne, France).
- Sex: Female
- Age/weight at study initiation: 18-20 weeks; mean body weight of 3451 g (range: 2950 g to 4095 g).
- Number of animals per group:22
The animals were individually housed in stainless steel cages, in a barriered rabbit unit, under specific pathogen free (SPF) standard laboratory conditions.
. temperature : 18 ± 3°C
. relative humidity : 50 ± 20%
. light/dark cycle : 8h dark/16h light (5:00 - 21:00)
. ventilation : about 8 to 10 cycles/hour of filtered, non-recycled air.

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
- Concentration in vehicle: 0, 0.3, 1 and 3 mg test substance/mL The test item dosage forms were prepared daily and stored at room temperature prior to use.
- Chemical analysis: The concentration of samples taken from each control and test substance dosage form prepared for use on the first day of treatment and on the last day of treatment was determined.
- Total volume applied: The quantity of dosage form administered to each animal was adjusted according to the most recently recorded body weight. A constant dosage-volume of 10 mL/kg/day was used.
Analytical verification of doses or concentrations:
not specified
Details on mating procedure:
Animals were mated at the breeder's facilities. The day of confirmed mating (visual assessment) was designated as day 0 post-coitum (p.c.) or day 0 of gestation (GD 0). The animals were supplied to CIT on day 1 post-coitum; consequently they were acclimated for a period of 5 days before the beginning of the treatment period.
Duration of treatment / exposure:
Day 6 to 28 post coitum
Frequency of treatment:
Daily
Duration of test:
23 d
Dose / conc.:
0 mg/kg bw/day
Remarks:
vehicle alone
Dose / conc.:
3 mg/kg bw/day
Dose / conc.:
10 mg/kg bw/day
Dose / conc.:
30 mg/kg bw/day
No. of animals per sex per dose:
22 mated females per dose
Control animals:
yes, concurrent vehicle
Details on study design:
The dose-levels were selected following the results of a previously conducted range finding study (CIT/Study No. 26147RSL): no maternal toxic effects were recorded at 3, 10 or 30 mg a.s./kg/day, but marked toxicity was observed at 50 and 100 mg/kg/day, which resulted in the deaths of a high number of females in both high dosegroups. At these two latter dose-levels, clinical signs (orange coloured urine, soft faeces, soiled urogenital area, emaciated appearance…), body weight loss, lower food consumption and reddish colour of the stomach mucosa were observed in most females. Consequently, the dose-levels of 3, 10 and 30 mg/kg/day were selected for the main study.
Maternal examinations:
- Body weight Yes: each female on days 2, 4, 5, 6, 9, 12, 15, 18, 22, 26 and 29 post-coitum.
- Food consumption: The quantity of food consumed by each female was recorded for days 2-4, 4-5, 5-6, 6-9, 9-12, 12-15, 15-18, 18-22, 22-26 and 26-29 post-coitum. Any obvious spillage of food was documented.
- Clinical signs: Morbidity and mortality. From the start of the treatment period, each animal was observed at least once a day, at approximately the same daily time for the recording of clinical signs (including evidence of abortion/desorption).



Ovaries and uterine content:
- On Day 29 post-coitum, all the surviving dams were sacrificed and subjected to a macroscopic post-mortem examination. A gross examination of placentas was also performed. The fetuses were removed by hysterectomy and the uterus weighed. The net body weight gain was calculated. The litter parameters, namely, the number of corpora lutea, implantation sites, early and late resorptions, dead and live fetuses were recorded. The fetuses were weighed and submitted to external examination.
Fetal examinations:
- The live fetuses were killed and then subjected to a fresh dissection and detailed examination of soft tissue, including body, head and brain. The sex was determined. The carcasses were then fixed and the skeletons (including cartilage) stained and examined.

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
At 30 mg/kg bw/day:
- the relevant clinical signs concerned the deceased females and two prematurely sacrificed females. No other clinical signs were noted in females from this group.
At 10 mg/kg bw/day:
- there were no relevant clinical signs except for two females with blood in the bedding on Days 22 and 23 post-coitum or absence of feces from Day 26 post-coitum
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, treatment-related
Description (incidence):
At 30 mg/kg bw/day:
- three females died and two females were prematurely sacrificed for ethical reasons or abortion.
At 10 mg/kg bw/day:
- there were no deaths.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 30 mg/kg bw/day:
- body weight gain was transiently reduced (GD 9-12, -70% below the control, p<0.05) but returned to normal values thereafter.
At 10 mg/kg bw/day:
- reduction of maternal body weight gain did not reach statistical significance.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
At 10 mg/kg bw/day:
- reduction of food consumption did not reach statistical significance.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Description (incidence and severity):
At 30 mg/kg bw/day:
- the necropsies revealed in 8/22 females: accentuated lobular pattern in the liver, pale liver, whitish areas and/or blackish deposits and/or edema in the stomach mucosa, reddish or brownish foci on the lungs, blackish contents in the intestines, dilated intestines and dilated gall bladder.
At 10 mg/kg bw/day:
- the necropsies revealed in 5/22 females: dilated gall bladder, accentuated lobular pattern.
- pale liver, brownish or reddish foci on the lungs, blackish deposit on the stomach mucosa.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
no effects observed
Details on results:
At 30 mg/kg/day:
- three females died and two females were prematurely sacrificed for ethical reasons or abortion,
- the relevant clinical signs concerned the deceased females and two prematurely sacrificed females. No other clinical signs were noted in females from this group.
- body weight gain was transiently reduced (GD 9-12, -70% below the control, p<0.05) but returned to normal values thereafter,
- the necropsies revealed in 8/22 females: accentuated lobular pattern in the liver, pale liver, whitish areas and/or blackish deposits and/or oedema in the stomach mucosa, reddish or brownish foci on the lungs, blackish contents in the intestines, dilated intestines and dilated gall bladder,

At 10 mg/kg/day:
- there were no deaths. Reduction of maternal body weight gain and food consumption did not reach statistical significance,
- there were no relevant clinical signs except for two females with blood in the bedding on days 22 and 23 post-coitum or absence of faeces from day 26 post- coitum,
- the necropsies revealed in 5/22 females: dilated gall bladder, accentuated lobular pattern, - pale liver, brownish or reddish foci on the lungs, blackish deposit on the stomach mucosa,

At 3 mg/kg/day:
no signs of maternal toxicity.
Number of abortions:
no effects observed
Pre- and post-implantation loss:
effects observed, non-treatment-related
Description (incidence and severity):
The fluctuations recorded for the mean number of resorptions (early or late) and the mean number of dead foetuses and consequently for the percentages of post-implantation loss were also not considered to be treatment-related, as they were minimal and not dose-related.
Total litter losses by resorption:
no effects observed
Early or late resorptions:
effects observed, non-treatment-related
Description (incidence and severity):
The fluctuations recorded for the mean number of resorptions (early or late) and the mean number of dead foetuses and consequently for the percentages of post-implantation loss were also not considered to be treatment-related, as they were minimal and not dose-related.
Dead fetuses:
effects observed, non-treatment-related
Description (incidence and severity):
The fluctuations recorded for the mean number of resorptions (early or late) and the mean number of dead foetuses and consequently for the percentages of post-implantation loss were also not considered to be treatment-related, as they were minimal and not dose-related.
Changes in pregnancy duration:
not examined
Changes in number of pregnant:
not examined
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
The fluctuations noted in the mean number of corpora lutea and implantation sites were slight and not dose-related, they were consequently considered not to be treatment-related.
Details on maternal toxic effects:
At 3 mg/kg bw/day:
- no signs of maternal toxicity
Key result
Dose descriptor:
NOAEL
Remarks:
systemic toxicity
Effect level:
3 mg/kg bw/day
Based on:
act. ingr.
Basis for effect level:
gross pathology
other: based on dilated gall bladder, accentuated lobular pattern in liver, transient but severe body weight gain reduction on GD 9-12 and mortality at top dose
Remarks on result:
other: Blackish content in stomach and intestines is indicative of local corrosive effects of test substance related to the dosing of substance by gavage
Key result
Abnormalities:
effects observed, treatment-related
Localisation:
other: general toxicity, no effects observed on reproductive organs
Fetal body weight changes:
no effects observed
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
no effects observed
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
The occurrence of some external malformations throughout all treated groups, including the controls, did not suggest any substance-related origin because of their low incidence, absence of dose-relationship and/or statistical significance.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
No relevant malformations was noted but the presence of a full supernumerary 13th pair of ribs (as a foetal variation), was markedly increased at 10 and 30 mg/kg bw/day. These differences in foetal or litter incidence, which were within background data, were most probably due to a low control value. The incidence of one other skeletal variation (unossified 5th sternebra) was significantly greater but in the low dose-group only.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
The occurrence of some soft tissue malformations throughout all treated groups, including the controls, did not suggest any substance-related origin because of their low incidence, absence of dose-relationship and/or statistical significance.
Details on embryotoxic / teratogenic effects:
Conclusion: 
There were no treatment-related effects on litter data parameters and no treatment-related findings upon external, visceral or skeletal observation in any of the dose groups. The test substance was not teratogenic in rabbits.
Key result
Dose descriptor:
NOAEL
Remarks:
general and developmental toxicity
Effect level:
30 mg/kg bw/day
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: No treatment related developmental effect up to the highest dose
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no

Result: 

Range-finding study:

Groups of six mated female rabbits received daily oral administration at 0 (vehicle, purified water), 3, 10 or 30 mg C12 -16 ADBAC/kg bw/day from day 6 to day 28 post-coitum. In the absence of maternal toxicity, additional groups with dose levels 0, 50 and 100 mg C12 -16 ADBAC/kg bw/day were added.

C12 -16 ADBAC from Day 6-28 p.c. produced marked maternal toxicity at 50 and 100 mg/kg/day shown by high number of deceased females and severe clinical conditions in most animals.

At 30 mg/kg bw/day, slightly high mean number of post-implantation loss associated with slightly low mean number of live foetuses were recorded. No maternal toxicity or effects on litter data parameters were noted at 3 and 10 mg/kg/day.

Based on these results, the 30 mg/kg bw/day dose-level was chosen as the high dose-level in the further developmental toxicity study (CIT Study No. 26148 RSL).

Main study:

The test substance was not teratogenic in rabbits.

- LO(A)EL maternal toxic effects:
10 mg/kg bw/d, based on necropsy findings in 5/22 animals. Incidence was increased to 8/22 at 30 mg/kg bw/day (dilated gallbladder 3/22, accentuated lobular pattern liver 3/22).
Foci (reddish/brownish) in the lung was also observed in 2 females, but also in view of findings in range finding study and parallel study with comparable compound, this can be caused by inadvertent presence of substance into the airways and not attributable to systemic toxicity. Incidence was not increased in the top-dose group. There is an indication of lower body weight gain, correlating to a lower food consumption, but that was not statistical significant and in the high-dose goup not different from the mid-dose group. Blackish content in stomack and intestines is indicative of local corrosive effects of test substance.

- NO(A)EL maternal toxic effects:
3 mg/kg bw/day. There seems to be a dose-response related increase in necropsy findings in stomach, intestine and liver. Dilated gallbladder incidence was not increased in highest dose group compared to mid-dose.

- LO(A)EL embryotoxic / teratogenic effects:
Based on the number of foetuses presenting malformations or variations and in the absence of a treatment-related increase of such observation, the embryo-fetal development was not considered to be affected by treatment.
- NO(A)EL embryotoxic / teratogenic effects:
30 mg/kg bw/day, being the highest tested dose.

Conclusions:
Under the study conditions, the NOAEL for maternal toxicity and embryo-foetal development was established at 3 and 30 mg a.i./kg bw/day respectively in rabbits.
Executive summary:

A study was conducted to determine the developmental toxicity and teratogenicity of the test substance, C12-16 ADBAC (49.9% active in water), according to OECD Guideline 414 and US EPA OPPTS 870.3700, in compliance with GLP. The substance was administered to pregnant rabbits by gavage from Day 6 to 28 post-coitum at the dose-levels of 0, 3, 10 or 30 mg a.i./kg bw/day. Based on the results of a range-finding study, the test subsatnce was administered as a solution by daily oral administration at 3, 10 and 30 mg a.i./kg/day from day 6 to day 28 post-coitum. One group of 22 females received the vehicle alone (purified water) under the same experimental conditions and acted as a control group. A standard dosage-volume of 10 mL/kg body weight was used for each group. Food consumption and body weight were recorded daily throughout the study period. Clinical signs were checked each day. On day 29 post-coitum, all the surviving dams were sacrificed and subjected to a macroscopic post-mortem examination. The foetuses were removed by hysterectomy and the uterus weighed. The net body weight gain was calculated. The litter parameters, namely, the number of corpora lutea, implantation sites, early and late resorptions, dead and live foetuses were recorded. The foetuses were weighed and submitted to external examination. The live fetuses were killed and then subjected to a fresh dissection and detailed examination of soft tissue, including body, head and brain. The sex was determined. The carcasses were then fixed and the skeletons (including cartilage) stained and examined. The dose of 30 mg a.i./kg bw/day caused the death of three females, severe clinical condition or abortion in two other females and transient, lower maternal body weight gain. Necropsies revealed in 8/22 females accentuated lobular patterns in the liver, whitish areas and/or blackish deposits and/or oedema in the stomach mucosa, reddish or brownish foci on the lungs, blackish contents in the intestines, dilated intestines and dilated gall bladder. At 10 mg/kg bw/day, also an indication of corresponding lower food consumption was observed. Relevant necropsy findings were noted in 5/22 females ((dilated gall bladder, accentuated lobular pattern, pale liver, brownish or reddish foci on the lungs, blackish deposit on the stomach mucosa). Blackish content in stomach and intestines was considered to be indicative of local corrosive effects of the test substance. No maternal toxicity or effects on litter data parameters or embryo-foetal development were noted at 3 mg/kg bw/day. Moreover, there were no effects on litter data parameters and no treatment-related findings upon external, visceral or skeletal observation in any of the dose groups up to 30 mg a.i./kg bw/day. Under the study conditions, the NOAEL for maternal toxicity and embryo-foetal development was established at 3 and 30 mg a.i./kg bw/day respectively in rabbits (Gaoua, 2005).

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1992
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
(Test substance was administered from Days 6-15 of gestation; however, guideline recommends dosing from implantation through the entire period of gestation to the day before caesarean section.)
Qualifier:
equivalent or similar to guideline
Guideline:
EPA OPP 83-3 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
(Test substance was administered from Days 6-15 of gestation; however, guideline recommends dosing from implantation through the entire period of gestation to the day before caesarean section.)
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on test animals or test system and environmental conditions:
Test animals:
- Source: Sprague Dawley CD rats (Crl: CD BR) were obtained from Charles River Breeding Laboratories, Portage MI USA.
- Age at study initiation: 11 weeks
- Weight at study initiation: 245.0-246.6 g
- Housing: Animals were housed singly in stainless steel cage, wire mesh caging; dimension, 22.5 x 15.5 x 18.0 cm during the study.
- Diet: Ground Certified Rodent Chow # 5002 (Ralston Purina Company, St. Louis, MO), ad libitum
- Water: Tap water, ad libitum. Water was provided by an automatic watering system with demand control valves mounted on each rack.
- Acclimation period: 2 weeks

Environmental conditions:
- Temperature: 66-77°F
- Humidity: 40-70%
- Air changes: 8/h

In-life dates: From: 14 October 1991 to: 29 November 1991
Route of administration:
oral: gavage
Vehicle:
water
Remarks:
(Milli-Q water)
Details on exposure:
Preparations of dosing solutions: Dosing solutions were prepared by dissolving appropriate amount of the test substance with Milli-Q water. Concentrations were adjusted for percent active ingredient of the test substance.
- Rate of dose preparation: Dosing solutions were prepared once.
- Storage of dose formulations: Room temperature

Vehicle
- Concentration in vehicle: 0, 2, 6, and 20 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg bw/day

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dose formulations were analysed for concentration, homogeneity and stability by HPLC method.
- The homogeneity study performed on samples from the 10 and 100 mg/kg bw/day concentrations indicated that the test substance was uniformly distributed in the solution.
- The stability study conducted on lowest and highest concentrations indicated that the test substance was stable in the solutions for at least 14d in a glass flask when stored at room temperature.
- Concentration verification analyses of the dosing solutions showed analytical mean values ranging from 94.5 to 104.8% of nominal for all 3 concentrations.
Details on mating procedure:
- Impregnation procedure: Co-housed
- If cohoused: Animals were co-housed in stainless steel wire mesh cages (30.5 x 31.0 x 18.0 cm).
- M/F ratio per cage: 1:1
- Length of cohabitation: Animals were cohoused until evidence of copulation was observed
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility: Each male was paired only once in the study.
- Verification of same strain and source of both sexes: Male and female rats were obtained from same source
- Proof of pregnancy: Presence of vaginal copulation plug. This day was designated as Day 0 of gestation
Duration of treatment / exposure:
From Day 6-15 of gestation
Frequency of treatment:
Once daily during exposure period
Duration of test:
16d (From Day 6 to Day 21 post coitum)
Dose / conc.:
0 mg/kg bw/day
Remarks:
vehicle alone
Dose / conc.:
10 mg/kg bw/day
Dose / conc.:
30 mg/kg bw/day
Dose / conc.:
100 mg/kg bw/day
No. of animals per sex per dose:
25
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dosages were selected by the study sponsor on the basis of a range finding study with the test substance (BBRC Project Report 54-613).
- Rationale for animal assignment: Immediately after mating the female rats were randomly allocated to treatment groups and control group using a stratified randomization program based on body weight on gestation Day 0.
- Animal assignment: Mated rats were assigned to the following groups.
Group 1 (vehicle control): 0 mg/kg bw/day
Group 2 (Low dose): 10 mg/kg bw/day
Group 3 (Mid dose): 30 mg/kg bw/day
Group 4 (High dose): 100 mg/kg bw/day
Maternal examinations:
Mortality: Yes
Time schedule: Twice daily

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Twice daily

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were recorded on Days 0, 6, 9, 12, 15, 18 and 21.

FOOD CONSUMPTION: Yes
- Time schedule: Food consumption was recorded for every 3 d intervals from Days 0 to 21 post coitum (Days 0-3, 3-6, 6-9, 9-12, 12-15, 15-18 and 18-21)

WATER CONSUMPTION AND COMPOUND INTAKE: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on: Day 21 post coitum
- The females were killed by CO2 asphyxiation and the fetuses removed by caesarean section.
- Organs examined: Gravid uterus, ovaries, cervix, vagina, and peritoneal and thoracic cavities

OTHER: Liver and gravid uterine were weighed.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
- The fetuses were removed from the uterus, sexed, weighed individually and examined for variations and malformations including cleft palate
- External examinations: Yes (all per litter)
- Soft tissue examinations: Yes (approximately half per litter)
- Skeletal examinations: Yes (approximately half per litter)
- Head examinations: Yes (approximately half per litter)
- Body weight: Yes
Statistics:
- Levene’s test for equal variances, analysis of variance, and a pooled t-tests for pairwise comparisons.
- Nonparametric data were analyzed with Kruskal-Wallis test followed by Mann-Whitney U test when appropriate.
- Incidence data were compared using Fisher’s Exact Test.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Perioral wetness in 67% of dams and audible respiration in 3 dams of high dose group. One dam in this group exhibited dehydration, unkempt appearance, loose feces, urine stains, and perioral wetness. Audible respiration in 2 dams of mid dose group. One of these dams also exhibited urine stains, gasping, perinasal encrustation, loose feces and perioral wetness.



Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There were no effects of treatment on gestational body weight and body weight gain in any dose group.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food consumption was reduced between Days 6 to 9 in the mid and high dose group.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
There were no effects of treatment on the gravid uterine weight in any dose group.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Ulceration of stomach and gas-filled intestines, color changes in liver and lymph nodes and small spleen was seen in one dam of high dose group. Swollen liver was observed in one dam of mid dose group.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
not examined
Changes in number of pregnant:
not examined
Details on maternal toxic effects:
- Maternal toxic effects: Yes
- Developmental toxicity: There were no treatment-related differences in the number of ovarian corpora lutea and in gestational parameters including total number of implantations, number of viable and nonviable implants.
Key result
Dose descriptor:
NOEL
Remarks:
systemic toxicity
Effect level:
ca. 10 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
gross pathology
other: clinical signs and reduced food consumption
Remarks on result:
other: equivalent to 8.1 mg a.i./kg bw/day
Key result
Abnormalities:
effects observed, treatment-related
Localisation:
other: general toxicity
Description (incidence and severity):
Clinical signs, food consumption and gross pathological findings
Fetal body weight changes:
no effects observed
Description (incidence and severity):
No treatment-related effects on fetal body weights were observed in any group.
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
not examined
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Description (incidence and severity):
No test substance-related relevant effects during the external examination of fetuses were noted in any group.
Skeletal malformations:
no effects observed
Description (incidence and severity):
No treatment-related variations or malformations.
Visceral malformations:
no effects observed
Description (incidence and severity):
No treatment-related variations or malformations.
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects: No effects
Key result
Dose descriptor:
NOEL
Remarks:
general and developmental toxicity
Effect level:
ca. 100 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no effects observed
Remarks on result:
other: equivalent to 81 mg a.i./kg bw/day
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no
Conclusions:
Under the study conditions, the NOEL for maternal toxicity and development toxicity was established at 10 and 100 mg a.i./kg bw/day respectively (equivalent to 8.1 and 81 mg a.i./kg bw/day respectively).
Executive summary:

A study was conducted to determine the developmental toxicity and teratogenicity of the test substance, C12-16 ADBAC (81.09% active in aqueous/ethanol solution), according to OECD Guideline 414 and US EPA OPP 93 -3, in compliance with GLP. The experiment was performed in pregnant Sprague-Dawley CD rats. The test substance was administered to groups of 25 pregnant rats orally by gavage at dose levels of 0, 10, 30 and 100 mg/kg bw/day, once daily from Days 6 to 15 of gestation inclusive. Control animals were treated with the vehicle alone (Milli-Q water). Clinical observations were made twice daily, and maternal body weights were measured on gestation Days 0, 6, 9, 12, 15, 18 and 21. Maternal food consumption was measured at 3-day intervals from Day 0 to Day 21. All surviving females were sacrificed on Day 21 and the foetuses were examined for visceral and skeletal variations and malformations. No mortality was observed during the study. Treatment-related clinical signs included perioral wetness and audible respiration (in 3 dams) in high dose group. Audible respiration was also observed in 2 dams in the mid dose group. One of these dams also exhibited urine stains, gasping, perinasal encrustation, loose feces and perioral wetness. Food consumption was reduced between Days 6 to 9 in mid and high dose groups. There were no effects of treatment on gestational body weight and body weight gain and gravid uterine weight in any dose group. There were also no treatment-related differences in gestational parameters including total number of implantations, number of viable and nonviable implants between the control and the test groups. Further, there were no effects of treatment on fetal body weights per litter, or on the incidences of external, visceral and skeletal malformations and variations. Based on the results of the study, administration of the test substance by gavage to pregnant rats resulted in maternal toxicity at 100 mg a.i./kg bw/day. No developmental toxicity including teratogenicity was observed at any dosage employed. Under the study conditions, the NOEL for maternal toxicity and development toxicity was established at 10 and 100 mg a.i./kg bw/day respectively (equivalent to 8.1 and 81 mg a.i./kg bw/day respectively) (Neeper-Bradley, 1992).

Endpoint:
developmental toxicity
Type of information:
other: Literature data
Adequacy of study:
supporting study
Study period:
1985 and 1983
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
documentation insufficient for assessment
Principles of method if other than guideline:
Guideline not mentioned. Published data as weight of evidence.
GLP compliance:
not specified
Species:
other: rat and rabbit
Strain:
other: albino or Sprague-Dawley for rat and not specified for rabbit
Key result
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
50 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: no effects observed
Remarks on result:
other: oral route in rat
Key result
Dose descriptor:
NOAEL
Remarks:
reproductive
Effect level:
50 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: no effects observed
Remarks on result:
other: oral route in rat
Key result
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
30 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: no effects observed
Remarks on result:
other: oral route in rabbit
Key result
Dose descriptor:
NOAEL
Remarks:
reproductive
Effect level:
30 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: no effects observed
Remarks on result:
other: oral route in rabbit
Key result
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
150 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: no effects observed except for local skin reactions
Remarks on result:
other: dermal route in rat
Key result
Dose descriptor:
NOAEL
Remarks:
reproductive
Effect level:
150 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: no effects observed
Remarks on result:
other: dermal route in rat
Key result
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
50 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
gross pathology
Remarks on result:
other: vaginal route in rat
Key result
Dose descriptor:
NOAEL
Remarks:
reproductive
Effect level:
50 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
dead fetuses
early or late resorptions
total litter losses by resorption
other: decrease in foetal growth and pregnancy rate
Remarks on result:
other: vaginal route in rat
Key result
Abnormalities:
no effects observed
Description (incidence and severity):
except following vaginal administration
Key result
Dose descriptor:
NOAEL
Remarks:
general and developmental
Effect level:
50 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no effects observed
Remarks on result:
other: oral route in rat
Key result
Dose descriptor:
NOAEL
Remarks:
general and developmental
Effect level:
> 30 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no effects observed
Remarks on result:
other: oral route in rabbit
Key result
Dose descriptor:
NOAEL
Remarks:
general and developmental
Effect level:
150 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no effects observed
Remarks on result:
other: dermal route in rat
Key result
Dose descriptor:
NOAEL
Remarks:
general and developmental
Effect level:
50 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reduction in number of live offspring
fetal/pup body weight changes
changes in litter size and weights
skeletal malformations
Remarks on result:
other: vaginal route in rat
Key result
Abnormalities:
effects observed, treatment-related
Localisation:
skeletal: sternum
Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
100 mg/kg bw/day
Treatment related:
yes
Relation to maternal toxicity:
developmental effects occurring together with maternal toxicity effects, but not as a secondary non-specific consequence of maternal toxicity effects
Dose response relationship:
yes

Literature data:
-Oral:
Species: Pregnant albino rats
Exposure period: GD 6-15
Doses: 0, 10, 25 or 50 mg/kg bw/day stomach tube. Including a positive (aspirin) control.
Critical effects dams, fetuses: Among the treated groups, neither reproduction performance of the dam nor foetus weights differed from those of the control animals. The incidences of any skeletal abnormality and soft tissue abnormalities were no greater in the test groups than in the control groups. The incidence of both types of abnormalities was significantly greater in the aspirin-treated group. 
NO(A)EL Teratogenicity/Embryotoxicity: 50 mg/kg bw/day
(CIR Final Report on the Safety Assessment of Stearalkonium Chloride. 1977, cited by Bibra, 1989)

Species: pregnant rabbits
Exposure period: GD 7-19
Doses: 30 mg/kg bw/d or more of the test substance by gavage 
Critical effects dams, foetuses: maternal and embryo toxicity (unspecified) reported. No malformations were seen.
NO(A)EL Teratogenicity/Embryotoxicity: >30 mg/kg bw/day (CEC, 1987, cited in Bibra, 1989)

- Dermal:
Species: 20 mated, Sprague Dawley rats
Exposure period: GD 6-15
Doses: 0.5 mL 1.6, 3.3, 6.6% test substance was applied (uncovered) to the shaved skin of rats. (6.6% corresponds to
approximately 150 mg/kg bw/day)
Critical effects dams, fetuses: The doses induced local adverse maternal reaction (skin reactions), but not systemic
toxicity. No effects on litter size, post-implantation loss, litter and mean foetal weights were seen. No signs of
embryotoxicity or foetal abnormalities.

NO(A)EL Teratogenicity/Embryotoxicity: 150 mg/kg bw/day
NO(A)EL Maternal toxicity: 150 mg /kg bw/day (Palmer, 1983)

- Vaginal:
Species: Pregnant rats
Exposure period: Single dose on GD 1
Doses: 0, 25, 50, 100 and 200 mg/kg bw/day
Critical effects dams, fetuses: No adverse effects on pregnancy outcome at the lowest dose level. At 50 mg/kg bw/d
and above, there were decreases in the number of live pups per litter and in litter size and weight. No visceral
anomalies were seen, however abnormal bone development (sternal defects), increases in early embryo/foetal death
(resorptions), reduced foetal growth and slight decreases in pregnancy rate were seen at 100 mg/kg bw/day. In all rats given
100 mg/kg bw/day or more vaginal inflammation was seen at necropsy.
NO(A)EL Teratogenicity/Embryotoxicity: 50 mg/kg bw/day
NO(A)EL Maternal toxicity: 50 mg /kg bw/day (Buttar, 1985)

Conclusions:
Under the study conditions, the test substance presented developmental toxicity effects based on the study conducted in rats via vaginal route. The NOAEL for maternal, reproductive and developmental toxicity of the test substance was determined to 50 mg/kg bw/day.
Executive summary:

Studies were conducted to determine the developmental toxicity / teratogenicity of the test substance in rats and rabbits via oral, dermal and vaginal routes. Under the study conditions, the test substance presented maternal and developmental toxicity effects based on the study conducted in rats via vaginal route. The NOAEL for maternal, reproductive and developmental toxicity of the test substance was determined to 50 mg/kg bw/day (BIBRA, 1989, Buttar, 1985 and Palmer, 1983).

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
3 mg/kg bw/day
Study duration:
subacute
Experimental exposure time per week (hours/week):
168
Species:
rabbit
Quality of whole database:
Guideline compliant studies with read across substances
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

Study 1:A dose range-finding study was conducted to determine the doses needed for conducting developmental toxicity and teratogenicity stud with the test substance (49.9% active in water) according to OECD Guideline 414 (see below). Groups of six mated female rabbits received daily oral administration at 0 (vehicle, purified water), 3, 10 or 30 mg/kg bw/day from Day 6 to 28 post-coitum. In the total absence of maternal toxicity at all dose levels and to better define the top dose level of the main study, the study director suggested to perform a complementary study with higher dose levels of 50 and 100 mg/kg bw/day. The test substance from GD 6-28 produced marked maternal toxicity at 50 and 100 mg/kg bw/day shown by high number of deceased females and severe clinical conditions in most animals. At 30 mg/kg bw/day, a slightly higher mean number of post-implantation loss associated with a slightly lower mean number of live foetuses was recorded. No maternal toxicity or effects on litter data parameters were noted at 3 and 10 mg/kg bw/d. Based on these results, the 30 mg/kg bw/day dose level was chosen as the high dose level in the further developmental toxicity study (Gaoua, 2004).

Study 2:A study was conducted to determine the developmental toxicity and teratogenicity of the test substance, C12-16 ADBAC (49.9% active in water), according to OECD Guideline 414 and US EPA OPPTS 870.3700, in compliance with GLP. The substance was administered to pregnant rabbits by gavage from Day 6 to 28 post-coitum at the dose levels of 0, 3, 10 or 30 mg a.i./kg bw/day. Based on the results of a range-finding study, the test substance was administered as a solution by daily oral administration at 3, 10 and 30 mg a.i./kg/day from day 6 to day 28 post-coitum. One group of 22 females received the vehicle alone (purified water) under the same experimental conditions and acted as a control group. A standard dosage volume of 10 mL/kg body weight was used for each group. Food consumption and body weight were recorded daily throughout the study period. Clinical signs were checked each day. On day 29 post-coitum, all the surviving dams were sacrificed and subjected to a macroscopic post-mortem examination. The foetuses were removed by hysterectomy and the uterus was weighed. The net body weight gain was calculated. The litter parameters, namely, the number of corpora lutea, implantation sites, early and late resorptions, dead and live foetuses were recorded. The foetuses were weighed and submitted to external examination. The live fetuses were killed and then subjected to a fresh dissection and detailed examination of soft tissue, including body, head and brain. The sex was determined. The carcasses were then fixed and the skeletons (including cartilage) stained and examined. The dose of 30 mg a.i./kg bw/day caused the death of three females, severe clinical condition or abortion in two other females and transient, lower maternal body weight gain. Necropsies revealed in 8/22 females accentuated lobular patterns in the liver, whitish areas and/or blackish deposits and/or oedema in the stomach mucosa, reddish or brownish foci on the lungs, blackish contents in the intestines, dilated intestines and dilated gall bladder. At 10 mg/kg bw/day, also an indication of corresponding lower food consumption was observed. Relevant necropsy findings were noted in 5/22 females ((dilated gall bladder, accentuated lobular pattern, pale liver, brownish or reddish foci on the lungs, blackish deposit on the stomach mucosa). Blackish content in stomach and intestines was considered to be indicative of local corrosive effects of the test substance, which was related to the dosing of the substance by gavage. No maternal toxicity or effects on litter data parameters or embryo-foetal development were noted at 3 mg/kg bw/day. Moreover, there were no effects on litter data parameters and no treatment-related findings upon external, visceral or skeletal observation in any of the dose groups up to 30 mg a.i./kg bw/day. Under the conditions of the study, the NOAEL for maternal toxicity and embryo-foetal development was established at 3 and 30 mg a.i./kg bw/day respectively in rabbits (Gaoua, 2005).

Study 3:A study was conducted to determine the developmental toxicity and teratogenicity of the test substance, C12-16 ADBAC (81.09% active in aqueous/ethanol solution), according to OECD Guideline 414 and US EPA OPP 93 -3, in compliance with GLP. The experiment was performed in pregnant Sprague-Dawley CD rats. The test substance was administered to groups of 25 pregnant rats orally by gavage at dose levels of 0, 10, 30 and 100 mg/kg bw/day, once daily from Days 6 to 15 of gestation inclusive. Control animals were treated with the vehicle alone (Milli-Q water). Clinical observations were made twice daily, and maternal body weights were measured on gestation Days 0, 6, 9, 12, 15, 18 and 21. Maternal food consumption was measured at 3-day intervals from Day 0 to Day 21. All surviving females were sacrificed on Day 21 and the foetuses were examined for visceral and skeletal variations and malformations. No mortality was observed during the study. Treatment-related clinical signs included perioral wetness and audible respiration (in 3 dams) in high dose group. Audible respiration was also observed in 2 dams in the mid dose group. One of these dams also exhibited urine stains, gasping, perinasal encrustation, loose feces and perioral wetness. Food consumption was reduced between Days 6 to 9 in mid and high dose groups. There were no effects of treatment on gestational body weight and body weight gain and gravid uterine weight in any dose group. There were also no treatment-related differences in gestational parameters including total number of implantations, number of viable and nonviable implants between the control and the test groups. Further, there were no effects of treatment on fetal body weights per litter, or on the incidences of external, visceral and skeletal malformations and variations. Based on the results of the study, administration of the test substance by gavage to pregnant rats resulted in maternal toxicity at 100 mg a.i./kg bw/day. No developmental toxicity including teratogenicity was observed at any dosage employed. Under the study conditions, the NOEL for maternal toxicity and development toxicity was established at 10 and 100 mg/kg bw/day respectively (equivalent to 8.1 and 81 mg a.i./kg bw/day respectively) (Neeper-Bradley, 1992).

In addition, few more studies conducted with the test substance in rats and rabbits via oral, dermal and vaginal routes were identified from literature sources. Under the study conditions, the test substance presented maternal and developmental toxicity effects based on the study conducted in rats via the vaginal route. The NOAEL for maternal, reproductive and developmental toxicity of the test substance was determined to 50 mg/kg bw/day (BIBRA, 1989, Buttar, 1985 and Palmer, 1983).

The biocides assessment reports available from RMS Italy on C12-16 ADBAC and Coco TMAC (citing the above studies), concluded similarly that overall these substances do not indicate a concern for development toxicity at doses that are not toxic to the mother. Although there is of a bit a mix-up of the studies and NOAELs, which were summarized under Appendix 1 of the C12-16 ADBAC biocides assessment report, the maternal NOAELs (3 -10 mg/kg bw/day) which reflects local effects on the gut mucosa due to gavage bolus administration, were not considered to be relevant to the determination of a systemic AOEL (ECHA biocides assessment report, 2015, 2016).

Overall, the available oral and dermal pre-natal development toxicity studies with the test substances in rats and rabbits, indicate no concern for developmental toxicity. Furthermore, in line with the biocides assessment report, the maternal NOAELs from the pre-natal studies have not been considered further for risk assessment.

Justification for classification or non-classification

Based on the results of the two-generation reproductive toxicity and pre-natal development toxicity studies in rats and rabbits, the test substance does not warrant a classification according to the EU CLP criteria (Regulation 1272/2008/EC).​

Additional information