Benzododecinium chloride

Toxicity to reproduction

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1990

Report date:

1990

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

EPA OPP 83-4 (Reproduction and Fertility Effects)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Quaternary ammonium compounds, benzyl C12-C16 (even numbered)-alkyldimethyl chlorides

EC Number:

939-253-5

Molecular formula:

C12-16H25-33-(CH3)2-C6H5-N.CL

IUPAC Name:

Quaternary ammonium compounds, benzyl C12-C16 (even numbered)-alkyldimethyl chlorides

Constituent 2

Reference substance name:

Free amine + amine hydrochloride

IUPAC Name:

Free amine + amine hydrochloride

Test material form:

liquid

Test animals

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%

Administration / exposure

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

Doses / concentrations

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

Examinations

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

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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 / performance (P0)

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

Effect levels (P0)

1

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

2

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

Target system / organ toxicity (P0)

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

Results: P1 (second parental generation)

General toxicity (P1)

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 / performance (P1)

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

Effect levels (P1)

1

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

2

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

Target system / organ toxicity (P1)

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

2 000 ppm

System:

other: body weight

Treatment related:

yes

Dose response relationship:

yes

Results: F1 generation

General toxicity (F1)

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.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Details on results (F1)

NOEL (F1and F2 offspring) = 1000 ppm

Effect levels (F1)

1

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

2

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

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Results: F2 generation

General toxicity (F2)

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

Developmental neurotoxicity (F2)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F2)

Developmental immunotoxicity:

not examined

Details on results (F2)

NOEL (F1and F2 offspring) = 1000 ppm

Effect levels (F2)

1

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

2

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

Target system / organ toxicity (F2)

Key result

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Applicant's summary and conclusion

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).