Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

In the first key study, the test material was administered orally (via gavage) on a daily basis beginning 14 days before a 14 day cohabitation period. Dosage continued through the day prior to sacrifice, after completion of the cohabitation period (minimum of 39 days of administration). In female rats, dosage continued up to and including day 4 of lactation. Male and female rats assigned to the satellite recovery portion of the study were not administered the test substance and/or the vehicle for 14 days prior to sacrifice starting when the first main study female rat assigned to the main study reached day 4 of lactation.

Animals were observed regularly for body weight, mortality, clinical signs, food consumption, and behaviour. At the end of the study, the animals were sacrificed and examined for gross pathology, organ weights, histopathology, clinical chemistry, hematology, and urinalysis. In addition, each litter was evaluated for viability at least twice daily and clinical observations once daily. Each litter was counted once daily. Pup weights were recorded on lactation days 1 and 4 for litters of dams assigned to the main study haematology and clinical chemistry portion of the study. Pup body weights were also recorded periodically for various purposes. There were no significant effects to reproductive parameters. The NOAEL for reproduction in rats is 1000 mg/kg/day.  Additional details on the results:

Parental animals: No treatment-related mortalities were observed. Body weight gains of male rats only were significantly reduced in the 1000 mg/kg/day dosage group only.

Reproduction: No effects were observed on estrous, mating, fertility, natural delivery or litter observations at any dose. Pregnancy occurred in 10, 8, 10 and 10 female rats in the four respective dosage groups. All male rats with the exception of one in the 250 mg/kg/day dosage group mated successfully.

The second key study examined the effects of exposure to HAB component LAB on reproductive and developmental toxicity (read across).  In a two-generation reproductive study conducted under GLP conditions, CD rats were given a single daily dose via gastric intubation of 5, 50 or 500 mg/kg-bw/day over a 35-week period.  Clear evidence of toxicity was observed at the 500 mg/kg-bw/day dose level, with the most consistent effects being depressed weight gains in adults, smaller litters, fewer live pups, and decreased pup survival.  At 50 mg/kg-bw/day, the only effect was a temporary reduction in pup weight gain at day 7 that returned to normal at days 14 and 21.  This temporary reduction occurred in one generation only, and thus was not consistent across generations.  Therefore, given the significant effects at 500 mg/kg-bw/day and the non consistent effects at the lower dose, the NOAEL for two-generation reproductive toxicity of HAB component LAB (read across) is 50 mg/kg-bw/day for both parental and neonatal animals.

Link to relevant study records

Referenceopen allclose all

Endpoint:
toxicity to reproduction
Remarks:
other: OECD Guideline 422 Combined Study
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study.
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes
Species:
rat
Strain:
other: Crl:CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
Housing and Husbandry: F0 generation rats assigned to the main
satellite/recovery mated portions of the study were individually
housed in stainless steel, wire-bottomed cages, except during
the cohabitation and postpartum periods. During cohabitation, each
pair of male and female rats was housed in the male rat's cage.
Beginning no later than DG20, F0 generation female rats assigned
to natural delivery were individually housed in nesting boxes.
The study room was maintained under conditions of positive airflow,
Temperature and humidity were monitored and maintained. Animals
were given standard diet and water ad libitum under a 12 hour
light and 12 hour dark photoperiod.
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
Method Overview: Sixty male and 80 female rats were randomly assigned to four dosage groups, 15 male rates and 20 female rats per dosage group. The test material was administered orally (via gavage) on a daily basis beginning 14 days before a 14 day cohabitation period. Dosage continued through the day prior to sacrifice, after completion of the cohabitation period (minimum of 39 days of administration). In female rats, dosage continued up to and including day 4 of lactation. Male and female rats assigned to the satellite recovery portion of the study were not administered the test substance and/or the vehicle for 14 days prior to sacrifice starting when the first main study female rat assigned to the main study reached day 4 of lactation.

Dosage Selection: Doses were selected on the basis of results from a range-finding study. In that study there were no mortalities at doses as high as 2000 mg/kg/day. Body weight gains of the males only were reduced in the 1000 and 2000 mg/kg/day groups during the first week, and although they subsequently rebounded, the early reductions resulted in overall reduction at the end of the test period. Therefore, doses of 0, 250, 500 and 100 mg/kg/day were selected for the definitive test.

Details on mating procedure:
During cohabitation, each pair of male and female rats was housed in the male rat's cage.
Duration of treatment / exposure:
at least 39 days (males); through lactation day 4 (females)
Remarks:
Doses / Concentrations:
0 (corn oil vehicle), 250, 500 and 1000 mg/kg/day
Basis:

No. of animals per sex per dose:
15 male rates and 20 female rats per dosage group
Control animals:
yes, concurrent vehicle
Details on study design:
Stock Preparation: A blended stock suspension was prepared once at
the testing facility and stored at room temperature, protected
from light. Suspensions of the blended test substance and/or
vehicle for dosage administration were prepared daily and also
stored at room temperature, protected from light. Prepared
formulations were stirred continuously during dosage administration.
Parental animals: Observations and examinations:
Parameters Evaluated: All of the main and recovery rats were examined for viability, clinical observations, detailed clinical observations (main study rats only), body weight and body weight changes and feed consumption values. Maternal behavior was observed at various times for specific purposes. In addition, each litter was evaluated for viability at least twice daily and clinical observations once daily. Each litter was counted once daily. Pup weights were recorded on lactation days (DL) 1 and 4 for litters of dams assigned to the main study hematology and clinical chemistry portion of the study. Pup body weights were also recorded periodically for various purposes. Urine and blood samples were collected for evaluation periodically and a functional observational battery (FOB) and motor activity assessment was conducted on study day (DS) 40 (male rats) or DL5 (female rats). All surviving main study rats were sacrificed on the day following the last administration of the test substance and/or the vehicle, after a minimum of 39 days of dosage. Satellite/recovery mated female rats were sacrificed on DL18. Satellite/recovery non-mated male and female rats were sacrificed 4 days after the first main study rat reached DL4.
Postmortem examinations (parental animals):
Necropsy: The following tissues were weighed and/or retained from all rats: liver, kidneys, testes, epididymides, seminal vesicles with coagulation gland and prostate (male only), and ovaries, vagina, a mammary gland and uterus with cervix (female only). The following tissues were retained from the five male and female rats (main study) assigned to hematology, clinical biochemistry and histological evaluations: small and large intestines (Peyer's patches), lungs, trachea, esophagus, mandibular and mesenteric lymph nodes, peripheral nerve (sciatic), stomach, seminal vesicles with coagulating gland, spinal cord, thyroid, urinary bladder, prostate, vagina and a mammary gland, bone marrow and gross lesions. Additionally, the brain, adrenals, spleen, thymus and heart were weighed and retained for possible histological evaluation from the five male and female rats (main study) assigned to hematology, clinical biochemistry and histological evaluations. All gross lesions were examined histologically. Histological examination was performed on all (main study rats assigned to clinical chemistry evaluation only) control and high test substance dose group rats, and on the thymus and thyroids from all in the 250, 500 and 1000 mg/kg/day dosage groups. Pups that died were examined and/or preserved for future analysis. On DL4, pups from litters of dams assigned to the clinical chemistry and hematology portion of the study were sacrificed, as were pups from litters of dams assigned to the FOB portion of the study on DL5. Necropsied were performed.
Clinical signs:
no effects observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Other effects:
not examined
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Non-Reproduction Effects: No treatment-related mortalities were
observed. Body weight gains of male rats only were significantly
reduced in the 1000 mg/kg/day dosage group only. See the
accompanying summary in the repeated dose toxicity section.

Reproduction: No effects were observed on estrous, mating,
fertility, natural delivery or litter observations at any dose.
Pregnancy occurred in 10, 8, 10 and 10 female rats in the four
respective dosage groups. All male rats with the exception of
one in the 250 mg/kg/day dosage group mated successfully.
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: Reproductive parameters
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed
No effects were observed on estrous, mating, fertility, natural delivery or litter observations at any dose.
Dose descriptor:
other: not reported
Generation:
other: Offspring not reported
Effect level:
0 other: Offspring not reported
Sex:
not specified
Basis for effect level:
other:
Remarks on result:
other: Offspring not reported
Reproductive effects observed:
not specified
Conclusions:
Based on the lack of significant effects on any of the reproduction parameters, the reproduction NOAEL is 1000 mg/kg/day.
Executive summary:

This study determined the effects of the test substance on reproductive parameters. Groups of rats were exposed by oral gavage to concentrations of 0, 250, 500 or 1000 mg/kg/day of test substance. Males were exposed daily for 39 days, and females were exposed daily through lactation day 4. Animals were observed regularly for body weight, mortality, clinical signs, food consumption, and behaviour. At the end of the study, the animals were sacrificed and examined for gross pathology, organ weights, histopathology, clinical chemistry, hematology, and urinalysis. In addition, each litter was evaluated for viability at least twice daily and clinical observations once daily. Each litter was counted once daily. Pup weights were recorded on lactation days 1 and 4 for litters of dams assigned to the main study hematology and clinical chemistry portion of the study. Pup body weights were also recorded periodically for various purposes. There were no significant effects to reproductive parameters. The NOAEL for reproduction in rats is 1000 mg/kg/day.

Endpoint:
two-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP study published in peer-reviewed journal.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Version / remarks:
EPA/TSCA
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories
- Housing: wire mesh cages
- Diet (e.g. ad libitum): Ralson Purina commercial laboratory feed, ad libitum
- Water (e.g. ad libitum): tap water, ad libitum


ENVIRONMENTAL CONDITIONS
- Temperature (°F): 65-79
- Humidity (%): 17-76
- Photoperiod (hrs dark / hrs light): 12 hrs light/12 hrs dark
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: 7 nights, if female was still not pregnant, she was moved to another male for an additional 7 nights, and then to a third male if needed
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged (how): individually
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
daily
Frequency of treatment:
F0: Treatment was started 10 weeks before mating. For males, dosing continued for 2 weeks after mating (total of 105 days). For females, dosing continued through lactation for a total of 127 treatment days.

F1: The F1 generation was treated similarly to the F0 generation, but were exposed beginning 11 weeks pre-mating.
Remarks:
Doses / Concentrations:
0, 5, 50 and 500 mg/kg
Basis:
actual ingested
No. of animals per sex per dose:
30 animals of each sex per dose
Control animals:
yes, concurrent vehicle
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly


BODY WEIGHT: Yes
- Time schedule for examinations: males - weekly, females - weekly before mating, days 0, 7, 14, and 20 of gestation, and days 0, 4, 14, and 21 of lactation


FOOD CONSUMPTION: weekly

Litter observations:
STANDARDISATION OF LITTERS
F1 pups were selected for mating at weaning, at least one pup per litter was selected for the adult F1 generation


PARAMETERS EXAMINED
The following parameters were examined in F1 and F2 offspring:
number and sex of pups on days 0, 4, 7, 14, and 21 of lactation, postnatal mortality and presence of gross anomalies daily, weights on days 0, 4, 7, 14, and 21 of lactation

GROSS EXAMINATION OF DEAD PUPS:
yes
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals 2 weeks after mating.
- Maternal animals: All surviving animals at weaning.


GROSS NECROPSY
- Gross necropsy consisted of examination for gross lesions


HISTOPATHOLOGY / ORGAN WEIGHTS
pituitary glands, testes and epididymides, prostate and seminal vesicles, vagina, uterus, ovaries, and gross lesions
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at weaning.
- These animals were subjected to postmortem examinations similar to parental animals above.
Reproductive indices:
mating index, pregnancy rate, fertility index
Offspring viability indices:
pup survival
Clinical signs:
effects observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Reproductive performance:
effects observed, treatment-related
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
No mortality attributed to treatment was observed.


BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Mean body weights of high-dose-group (500 mg/kg bw/d) males were significantly and consistently reduced {12 = 0.01) in the FO (since premating week); mean body weights of high-dose females were significantly decreased in the FO generation since the 9th week of premating until the first week of lactation (p = 0.05); body weight reduction was significant on day 20 of gestation in both generations (p = 0.01


REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
Mating, pregnancy and fertility rates were not influenced.


GROSS PATHOLOGY (PARENTAL ANIMALS)
No gross postmortem findings.

HISTOPATHOLOGY (PARENTAL ANIMALS)
No histopathological findings.

Dose descriptor:
NOEL
Effect level:
50 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: Maternal and paternal general toxicity was reduced weight gain in the high dose group. Reduced litter size in the high dose group.
Mortality / viability:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
VIABILITY (OFFSPRING)
Litter size: Fl and F2 : significant decrease at 500 mg/kg bw/d (p = 0.05 and 0.01 respectively);
pup viability index at birth: Fl: significant decrease at 50 and 500 mg/kg bd/d (p = 0.05) F2: significant decrease at 500 mg/kg bw/d (p = 0.05), since effects at 50 mg/kg bw/d was only noted in a single generation, these effects were not considered biologically significant; survival of pups at day 4 was significantly decreased at 500 mg/kg bw/d in the Fl and F2 litters; survival of pups at day 21 (related to day 4) was significantly decreased only in the F2 litter at 50 mg/kg bw/d (p = 0.05), since effects at 50 mg/kg bw/d was only noted in a single generation, these effects were not considered biologically significant

BODY WEIGHT (OFFSPRING)
mean pup weight:
Fl litters; significant reduction at 50 and 500 mg/kg bw/d at day 7 and only at 500 mg/kg bw/d at day 14 and 21 (p = 0.05), since the reduction in the 50 mg/kg bw/d group was only noted at day 7 and only in the F1 generation, this effect was not considered biologically significant
F2 litters: significant reduction at 500 mg/kg bw/d at day 14 and 21 (p = 0.05).


OTHER FINDINGS (OFFSPRING)
In the high-dose-group, gestation length was significantly increased for the F2 litter interval (22.4 d compared to 22.0 in controls).
Dose descriptor:
NOEL
Generation:
F1
Effect level:
50 mg/kg bw/day
Sex:
not specified
Basis for effect level:
other: Reduced survival and weight gain in high dose group. Sporadic occurrences at mid dose level.
Dose descriptor:
NOEL
Generation:
F2
Effect level:
50 mg/kg bw/day
Sex:
not specified
Basis for effect level:
other: Reduced survival and weight gain in the high dose group. Sporadic occurrences at mid dose level.
Reproductive effects observed:
not specified

Litter Size and Pup Survival Indices

Dose (mg/kg/day)

Litter size (mean)

Pup viability index at birth (%)

Pup survival days 0-4 (%)

Pup survival days 0-4 (%)1

Pup survival days 4-21 (%)

F1 litters

0

12.7

99.1

93.8

95.7

5

12.8

98.0

93.4

91.0

50

13.1

95.4

92.3

84.2

500

10.0

95.4

85.0

94.7

F2 litters

0

11.3

98.6

80.2

93.5

98.1

5

11.6

98.2

87.3

94.4

97.1

50

13.1

97.3

89.5

92.3

89.1

500

7.0

89.0

84.1

85.9

97.3

1Excludes data for litters in which all pups died during the Day 0-3 interval.

Mean Pup Weights (g)

Dose (mg/kg/day)

Day 0

Day 4- Precull

Day 4- Postcull

Day 7

Day 14

Day 21

F1 litters

0

6.0

8.4

8.4

13.2

27.0

42.7

5

5.9

8.1

8.1

12.9

27.3

42.1

50

5.8

7.5

7.6

11.4

25.1

39.6

500

5.8

8.1

8.1

11.4

23.5

37.7

F2 litters

0

5.8

8.1

8.3

13.9

27.0

40.5

5

5.8

8.5

8.5

14.4

26.9

42.0

50

6.0

8.1

8.1

13.4

25.3

39.5

500

5.8

8.1

8.1

12.1

22.3

34.6

Conclusions:
The NOEL for the maternal and paternal generation was 50 mg/kg bw/day and for the offspring generations.
Executive summary:

This study examined the effects of exposure to the test substance on reproduction. Groups of 30 female and 30 male rats were exposed to concentrations of 0, 5, 50 and 500 mg/kg day of test substance by oral gavage beginning ten weeks before mating. Animals were then mated. The resulting generation was also exposed to the test substance and mated. Exposure continued through the mating period, and in females through the gestation and lactational periods. All animals were sacrificed and necropsied after exposure. Pups not used for mating were sacrificed at weaning. During the study, animals were observed for clinical signs, mortality, and body weight. Pups were examined for viability and body weight gain. Reproductive indices were also calculated. The NOAEL for parental toxicity was 50 mg/kg/day and the LOAEL was 500 mg/kg/day based on reduced weight gain and litter size in the high dose group. The NOAEL for offspring was 50 mg/kg/day based on reduced body weight gain and survival in the high and mid-dose groups. The LOAEL for offspring was 50 mg/kg/day.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
Key study is a repeat dose toxicity study with reproduction/developmental toxicity screening, reliable without restriction. A second key study reports experimental data gathered in a repeat dose study using a read across compound, reliable with restrictions.
Additional information

Justification for selection of Effect on fertility via oral route:
Key study reports experimental results, reliable without restriction.

Effects on developmental toxicity

Description of key information

The first of two key studies is the OECD 422 combined repeated dose and reproductive/developmental toxicity screening test.  In addition to the repeated dose endpoints, the study determined the effects of the test substance on developmental parameters. Groups of rats were exposed by oral gavage to concentrations of 0, 250, 500 or 1000 mg/kg/day of test substance. Males were exposed daily for 39 days, and females were exposed daily through lactation day 4. Animals were observed regularly for body weight, mortality, clinical signs, food consumption, and behaviour. At the end of the study, the animals were sacrificed and examined for gross pathology, organ weights, histopathology, clinical chemistry, haematology, and urinalysis. In addition, each litter was evaluated for viability at least twice daily and clinical observations once daily. Each litter was counted once daily. Pup weights were recorded on lactation days 1 and 4 for litters of dams assigned to the main study haematology and clinical chemistry portion of the study. Pup body weights were also recorded periodically for various purposes. There were no significant effects to developmental parameters. There were also no maternal toxic effects. The NOAEL for developmental toxicity in rats is > 1000 mg/kg/day. The NOAEL for maternal toxicity was also 1000 mg/kg/day.  Additional details on the results:

Maternal effects: Non-Developmental Effects: No treatment-related mortalities were observed. Body weight gains of male rats only were significantly reduced in the 1000 mg/kg/day dosage group only. No reproductive effects were observed. See the accompanying summaries in the repeated dose and reproduction toxicity sections.

Embryotoxic/teratogenic effects: Developmental Effects: As described in the reproduction section, no effects were observed on any of the reproductive or developmental parameters at any of the doses tested. The number of pups found dead or presumed cannibalized on postpartum days 2-4 was significantly increased, with a concomitant significant decrease in the viability index in the 250 mg/kg/day dosage group. These were not considered treatment-related because they were not dosage dependent and did not occur in the rats assigned to the recovery group. The number of mated rats that were pregnant, delivered live litters, or had stillborn pups, pups dying postpartum days 1 or 4-5, were not significantly different in any of the treatment groups compared to the vehicle control group. The duration of gestation, number of implantation sites per litter, and gestation index were also not significantly different. No effects on any of the developmental parameters were observed.

The second study determined the developmental toxicity of the test substance using the standard OECD 414 developmental toxicity test. Groups of 24 pregnant female rats were exposed to 0, 400, 800, or 1600 mg/kg bw/day of the test substance during days 6-15 of gestation. Females were sacrificed on day 20 of gestation, and the uteri examined for live and dead fetuses, number of implants, number of resorptions, and number of corpora lutea. There was reduced body weight gain in middle and high dose females during days 6 -15 of gestation. No other treatment related adverse effects were observed. No treatment related embryotoxic or teratogenic effects were noted. The maternal NOAEL was 400 mg/kg bw/day, and the LOAEL was 800 mg/kg bw/day based on the reduced body weight gain. The NOAEL for developmental toxicity was 1600 mg/kg bw/day.

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Jan. 13, 1987-Feb. 14, 1987
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes (incl. certificate)
Limit test:
yes
Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc.
- Age at study initiation: 49 days
- Weight at study initiation: 167-237 g females
- Housing: individually in stainless steel cages, except for mating, indentified by metal ear tag
- Diet (e.g. ad libitum): Purina Certified Rodent Chow No. 5002
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: 14 days


ENVIRONMENTAL CONDITIONS
- Temperature (°F): 63-80
- Humidity (%): 40-79%
- Photoperiod (hrs dark / hrs light): 12 hrs light/12 hrs dark


IN-LIFE DATES: From: Jan. 13, 1987 To: Feb. 14, 1987
Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Suspensions were prepared weekly.

VEHICLE
0.5% aqueous CMC suspension
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of the dosing suspension were dissolved in tetrahydrofuran, then analyzed with liquid chromatography. Detection was by an Ultra-Violet Detector at 254 nm. Analyses were performed weekly.
Details on mating procedure:
- Impregnation procedure: cohoused
- M/F ratio per cage: 1/1
- Length of cohabitation: 13 nights
- Proof of pregnancy: sperm in vaginal smear referred to as day 0
Duration of treatment / exposure:
days 6-15 of gestation
Frequency of treatment:
daily
Duration of test:
animals were sacrificed on day 20 of gestation
No. of animals per sex per dose:
24 mated females per dose
Control animals:
yes, concurrent vehicle
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily
- Cage side observations checked: cursory observation


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice daily for pharmacologic and toxicologic signs; on days 0, 6, 10, 12, 15, and 20 of gestation detailed physical exams were performed


BODY WEIGHT: Yes
- Time schedule for examinations: days 0, 6, 10, 12, 15, and 20 of gestation


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
Recorded for the intervals days 0-6, 6-10, 10-15, and 15-20

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: brain, nasal cavity, paranasal sinuses, thoracic, abdominal, and pelvic cavities

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
- Other: live fetuses, dead fetuses
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: half per litter
Statistics:
The following techniques were used:
analysis of variance, Bartlett's test, ANOVA, Dunnett's test, Kruskal-Wallis, Summed Rank Test, Regression Analysis, Jonckheere's Statistic, Arc Sine, Chi-square, Fisher Exact Test, Bonferroni Inequality, Armitage's Test
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
In the middle and high dose groups, there was slightly reduced mean weight gain during gestational days 6-15. This reduced weight gains were not statistically significant. No other treatment related adverse effects were observed.
Dose descriptor:
NOAEL
Effect level:
400 mg/kg bw/day (nominal)
Basis for effect level:
other: maternal toxicity
Dose descriptor:
LOAEL
Effect level:
800 mg/kg bw/day (nominal)
Basis for effect level:
other: maternal toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
No treatment related embryotoxic or teratogenic effects were noted.
Dose descriptor:
NOAEL
Effect level:
> 1 600 mg/kg bw/day (nominal)
Sex:
not specified
Basis for effect level:
other: developmental toxicity
Abnormalities:
not specified
Developmental effects observed:
not specified

Maternal Body Weight Change During Days 6-15 of Gestation (g)

Concentration

Mean (g)

Standard Deviation

Number

0 mg/kg day

39

9

23

400 mg/kg day

36

8

23

800 mg/kg day

34

10

22

1600 mg/kg day

31

14

23

Conclusions:
The maternal NOAEL in rats was 400 mg/kg bw/day. The maternal LOAEL was 800 mg/kg bw/day based on reduced weight gain. The developmental NOAEL was 1600 mg/kg bw/day.
Executive summary:

This study determined the developmental toxicity of the test substance Therminol 55. Groups of 24 pregnant female rats were exposed to 0, 400, 800, or 1600 mg/kg bw/day of the test substance during days 6 -15 of gestation. Females were sacrificed on day 20 of gestation, and the uteri examined for live and dead fetuses, number of implants, number of resorptions, and number of corpora lutea. There was reduced body weight gain in middle and high dose females during days 6 -15 of gestation. The maternal NOAEL was 400 mg/kg bw/day, and the LOAEL was 800 mg/kg bw/day based on the reduced body weight gain. The NOAEL for developmental toxicity was 1600 mg/kg bw/day.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study.
Qualifier:
according to
Guideline:
other: OECD Guideline 422. Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test.
Principles of method if other than guideline:
Method Overview: Sixty male and 80 female rats were randomly assigned to four dosage groups, 15 male rates and 20 female rats per dosage group. The test material was administered orally (via gavage) on a daily basis beginning 14 days before a 14 day cohabitation period. Dosage continued through the day prior to
sacrifice, after completion of the cohabitation period (minimum
of 39 days of administration). In female rats, dosage continued up
to and including day 4 of lactation. Male and female rats assigned
to the satellite recovery portion of the study were not
administered the test substance and/or the vehicle for 14
days prior to sacrifice starting when the first main study
female rat assigned to the main study reached day 4 of lactation.
GLP compliance:
yes
Species:
rat
Strain:
other: Crl:CD(SD)
Details on test animals and environmental conditions:
Housing and Husbandry: F0 generation rats assigned to the main satellite/recovery mated portions of the study were individually housed in stainless steel, wire-bottomed cages, except during the cohabitation and postpartum periods. During cohabitation, each pair of male and female rats was housed in the male rat's cage. Beginning no later than gestation day (DG) 20, F0 generation female rats assigned to natural delivery were individually housed in nesting boxes. The study room was maintained under conditions of positive airflow, temperature and humidity were monitored and maintained. Animals were given standard diet and water ad libitum under a 12 hour light and 12 hour dark photoperiod.
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
Dosage Selection: Doses were selected on the basis of results from a range-finding study. In that study there were no mortalities at doses as high as 2000 mg/kg/day. Body weight gains of the males only were reduced in the 1000 and 2000 mg/kg/day groups during the first week, and although they subsequently rebounded, the early reductions resulted in overall reduction at the end of the test period. Therefore, doses of 0, 250, 500 and 1000 mg/kg/day were selected for the definitive test.
Details on mating procedure:
During cohabitation, each pair of male and female rats was housed in the male rat's cage.
Duration of treatment / exposure:
at least 39 days (males); through lactation day 4 (females)
Frequency of treatment:
daily
No. of animals per sex per dose:
15 male rates and 20 female rats per dosage group
Control animals:
yes, concurrent vehicle
Details on study design:
Stock Preparation: A blended stock suspension was prepared once at the testing facility and stored at room temperature, protected from light. Suspensions of the blended test substance and/or vehicle for dosage administration were prepared daily and also stored at room temperature, protected from light. Prepared formulations were stirred continuously during dosage administration.
Maternal examinations:
Parameters Evaluated: All of the main and recovery rats were examined for viability, clinical observations, detailed clinical observations (main study rats only), body weight and body weight changes and feed consumption values. Maternal behavior was observed at various times for specific purposes. Urine and blood samples were collected for evaluation periodically and a functional observational battery (FOB) and motor activity assessment was conducted on study day (DS) 40 (male rats) or lactation day (DL) 5 (female rats). All surviving main study rats were sacrificed on the day following the last administration of the test substance and/or the vehicle, after a minimum of 39 days of dosage. Satellite/recovery mated female rats were sacrificed on DL18. Satellite/recovery non-mated male and female rats were sacrificed 4 days after the first main study rat reached DL4.

Necropsy: The following tissues were weighed and/or retained from all rats: liver, kidneys, testes, epididymides, seminal vesicles with coagulation gland and prostate (male only), and ovaries, vagina, a mammary gland and uterus with cervix (female only). The following tissues were retained from the
five male and female rats (main study) assigned to hematology, clinical biochemistry and histological evaluations: small and large intestines (Peyer's patches), lungs, trachea, esophagus, mandibular and mesenteric lymph nodes, peripheral nerve (sciatic), stomach, seminal vesicles with coagulating gland, spinal cord, thyroid, urinary bladder, prostate, vagina and a mammary gland, bone marrow and gross lesions. Additionally, the brain, adrenals,
spleen, thymus and heart were weighed and retained for possible histological evaluation from the five male and female rats (main study) assigned to hematology, clinical biochemistry and histological evaluations. All gross lesions were examined histologically. Histological examination was performed on all (main study rats assigned to clinical chemistry evaluation only) control and high test substance dose group rats, and on the thymus and thyroids from all in the 250, 500 and 1000 mg/kg/day dosage groups.
Fetal examinations:
Each litter was evaluated for viability at least twice daily and clinical observations once daily. Each litter was counted once daily. Pup weights were recorded on DLs 1 and 4 for litters of dams assigned to the main study hematology and clinical chemistry portion of the study. Pup body weights were also recorded periodically for various purposes.

Pups that died were examined and/or preserved for future analysis. On DL4, pups from litters of dams assigned to the clinical chemistry and hematology portion of the study were sacrificed, as were pups from litters of dams assigned to the FOB portion of the study on DL5. Necropsies were performed.
Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
Non-Developmental Effects: No treatment-related mortalities were
observed. Body weight gains of male rats only were significantly
reduced in the 1000 mg/kg/day dosage group only. No reproductive
effects were observed. See the accompanying summaries in the
repeated dose and reproduction toxicity sections.
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Basis for effect level:
other: maternal toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Developmental Effects: As described in the reproduction section
above, no effects were observed on any of the reproductive or
developmental parameters at any of the doses tested. The
number of pups found dead or presumed cannibalized on
postpartum days 2-4 was significantly increased, with a
concomitant significant decrease in the viability index in
the 250 mg/kg/day dosage group. These were not considered
treatment-related because they were not dosage dependent and
did not occur in the rats assigned to the recovery group. The
number of mated rats that were pregnant, delivered live litters,
or had stillborn pups, pups dying postpartum days 1 or 4-5, were
not significantly different in any of the treatment groups
compared to the vehicle control group. The duration of gestation,
number of implantation sites per litter, and gestation index
were also not significantly different. No effects on any of the
developmental parameters were observed.
Dose descriptor:
NOAEL
Effect level:
> 1 000 mg/kg bw/day (nominal)
Sex:
not specified
Basis for effect level:
other: developmental toxicity
Abnormalities:
not specified
Developmental effects observed:
not specified
Conclusions:
Based on the lack of developmental effects, the developmental NOAEL is 1000 mg/kg/day. The NOAEL for maternal toxicity was also 1000 mg/kg/day.
Executive summary:

This study determined the effects of the test substance on developmental parameters. Groups of rats were exposed by oral gavage to concentrations of 0, 250, 500 or 1000 mg/kg/day of test substance. Males were exposed daily for 39 days, and females were exposed daily through lactation day 4. Animals were observed regularly for body weight, mortality, clinical signs, food consumption, and behaviour. At the end of the study, the animals were sacrificed and examined for gross pathology, organ weights, histopathology, clinical chemistry, hematology, and urinalysis. In addition, each litter was evaluated for viability at least twice daily and clinical observations once daily. Each litter was counted once daily. Pup weights were recorded on lactation days 1 and 4 for litters of dams assigned to the main study hematology and clinical chemistry portion of the study. Pup body weights were also recorded periodically for various purposes. There were no significant effects to developmental parameters. There were also no maternal toxic effects. The NOAEL for developmental toxicity in rats is > 1000 mg/kg/day. The NOAEL for maternal toxicity was also 1000 mg/kg/day.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 600 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Key study is a repeat dose toxicity study with reproduction/developmental toxicity screening, reliable without restriction. A second key study reports experimental data gathered in a repeat dose study using a read across compound, reliable with restrictions.
Additional information

Justification for selection of Effect on developmental toxicity: via oral route:
Key study reports experimental results, reliable without restriction.

Justification for classification or non-classification

Reproduction and developmental toxicity was examined in a combined repeated dose and reproductive/developmental toxicity screening study (OECD 422) and a standard developmental toxicity study (OECD 414). Results indicate NOAELs for both maternal and reproductive toxicity of 1000 mg/kg bw/d in the combined test. No treatment related mortalities or significant effects were observed in the parental animals. No effects on reproduction were observed up to and including the highest dose tested. Similarly, the NOAEL for both maternal and developmental toxicity was 1000 mg/kg bw/d in the same combined test. No treatment related adverse effects on any of the developmental parameters were observed in any dose. In the OECD 414 study, test doses ranged up to 1600 mg/kg bw/d. Reduced body weight gain in the dams in the middle and high doses was the effect responsible for the LOAEL of 800 mg/kg bw/d and the NOAEL of 400 mg/kg bw/d. No treatment related developmental effects were observed, so the NOAEL for developmental toxicity is 1600 mg/kg bw/d, the highest dose tested.

HAB elicits no reproductive or developmental effects up to and including the highest doses tested.