Phenol, para-alkylation products with C10-15 branched olefins (C12 rich) derived from propene oligomerization, reaction products with sulphur monochloride and dec-1-ene

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records

Reference

Endpoint:

two-generation reproductive toxicity

Remarks:

based on test type (migrated information)

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

18th March 1994 to 17th February 1995 (experimental phase)

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Guideline study following GLP. This study is being used as read across from Phenol, tetrapropenyl-, sulfurized, carbonates, calcium salts, overbased CAS No. 122384-86-5, therefore reliability is reduced to 2.

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

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc., Portage, Michigan
- Age at study initiation: The animals were approximately six weeks old at the initiation of test article administration.
- Weight at study initiation: Male body weights ranged from 146 g to 216 g and female body weights ranged from 116 g to 177 g on the day of randomization.
- Breeding: The F0 animals were approximately 16 weeks old. Prior to the Ft pairing (week 29), male body weights ranged from 362 g to 648 g and female body weights ranged from 202 g to 348 g.
The F1 animals were 14 to 16 weeks old. Prior to the Ft satellite pairing (week 30), male body weights ranged from 388 g to 610 g and female body weights ranged from 199 g to 394 g. The F1 satellite animals were approximately 15 to 17 weeks old.
- Housing:
Following the initial acclimation period (F0) or selection (F1 and F1 satellite) and until pairing, all F0, F1 and F1 satellite test animals were individually housed in clean, wire-mesh cages suspended above cage-board. The animals were paired for mating in the home cage of the male. Following positive evidence of mating, the males were housed in suspended wire-mesh cages until the scheduled necropsy of the parental generations and the females were transferred to plastic maternity cages with nesting material (Bed-O'Cobs; The Andersons, Industrial Products Division, Maumee, OH 43537). The dams were housed in these cages until weaning on lactation day 21. The F1 satellite dams were housed in these cages until necropsy on lactation day 7, Following weaning of the litters in the F1 and F2 generations, the females were individually housed in suspended wire-mesh cages until the scheduled necropsy. Females for which there was no evidence of mating were placed in a plastic maternity cage with nesting material upon completion of a 15-day mating period. If these animals did not deliver after 25 days, they were returned to individual suspended wire-mesh cages. Animals were housed in accordance with the National Institutes' of Health "Guide for the Care and Use of Laboratory Animals". The animal facilities at WIL Research Laboratories, Inc., are accredited by the American Association for Accreditation of Laboratory Animal Care (AAALAC).
- Diet (e.g. ad libitum): Ad libitum. The basal ration used in this study was Purina Certified Rodent Chowo #5002. The diet utilized at WIL Research Laboratories, Inc., was a certified feed with appropriate analyses performed and provided by the manufacturer.
- Water (e.g. ad libitum): Ad libitum. Municipal water supplying the facility is sampled for contaminants according to Standard Operating Procedures.
- Acclimation period: During the acclimation period (15 days), the animals were observed twice daily for mortality and moribundity.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 72° ± 4° F
- Humidity (%): 30-70%.
- Air changes (per hr): Air handling units were set to provide approximately 10 fresh air changes per hour.
- Photoperiod (hrs dark / hrs light): Light timers were calibrated to provide a 12-hour light/12-hour dark photoperiod.

Route of administration:

oral: gavage

Vehicle:

peanut oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
For the control group, the appropriate amount of vehicle control article, peanut oil, was dispensed weekly into a properly-labeled storage container. The storage container was wrapped in foil, and the control article was stirred continuously throughout the sampling and dosing procedures using a magnetic stir bar and plate.
An appropriate amount of the test article was drawn into a syringe for each treated group. A sufficient amount of the vehicle, peanut oil, was added to a properly-labeled storage container. The test article was slowly added to the vehicle while being mixed on a Polytron® PT 6000 until all of the test article was incorporated into the vehicle. The mixtures were stirred using a magnetic stir bar and plate throughout the sampling, dispensation and dosing procedures. The storage containers were wrapped in foil for additional protection from light. The preparations were allowed to stir for 30 to 45 minutes prior to sampling. A sufficient amount of dose formulation from each group was subdivided into seven containers and stored at room temperature. One container per group was dispensed daily and allowed to stir for approximately 30 to 45 minutes prior to dose administration.
Preparations for all dose groups were formulated weekly. The preparations were stored at room temperature, protected from light. Inspection of the dosing formulations by the study director or alternate principal investigator prior to initiation of dosing was inadvertently not performed. This deviation was determined to have no adverse effect on the outcome of the study.
On July 21 and 29, 1994 (week 18 and 19, respectively) excess amounts of the vehicle were inadvertently added to the Group 2, 3 and 4 formulations. However, these amounts were slight (5.4%, 0.7% and 1.6%, respectively), and no adverse effect on the outcome of the study was evident.

Dosing solutions in peanut oil vehicle were prepared weekly, and their test material concentration (weeks 0, 1, 2, 3, 7, 11, 24, and 37), homogeneity
and stability verified by chemical analysis. There were 30 rats/sex/group for the F0 and F1 generations. In addition there were F1 satellite control
(30/sex) and high-dose (28 males/29 females) groups used for cross-breeding and recovery evaluations. Male and female parental animals for each generation were dosed daily during the pre-mating (intervals shown above), mating (15 days), gestation (up to 25 days) and lactation (21 days)
periods until necropsy, except F1 satellite males were held for a 33-day recovery period before necropsy.

Details on mating procedure:

- M/F ratio per cage: F0 and F1 animals were paired within their groups on a 1:1 basis for mating. F1 satellite animals were cross-paired on a 1:1 basis, control males with high-dose females and vice versa.
- Length of cohabitation: When evidence of mating was not detected within 10 days, the female was placed for up to 5 days with another male from the same group that had previously mated.
- Proof of pregnancy: Females were examined daily during mating for presence of a copulatory plug or sperm in the vagina. The day when evidence of mating was identified was termed day 0 of gestation.
- After 10 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- After successful mating each pregnant female was caged (how): Following positive evidence of mating, the males were housed in suspended wire-mesh cages until the scheduled necropsy of the parental generations and the females were transferred to plastic maternity cages with nesting material (Bed-O'Cobs; The Andersons, Industrial Products Division, Maumee, OH 43537). The dams were housed in these cages until weaning on lactation day 21.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dosing solutions were made up weekly, and the test material concentration, homogeneity and stability were verified for weeks 0, 1, 2, 3, 7, 11, 24, and 37 by chemical analysis.

Duration of treatment / exposure:

Fo parental animals were dosed for 71 days prior to mating and throughout breeding, gestation and lactation until necropsy.
The offspring of the F0 generation (F1 litters) were potentially exposed to the test article in utero, through nursing during lactation days 0-21 and were administered the test article following weaning (beginning on day 22 post parturn). The F1 pups selected for breeding were administered the test article for a minimum of 77 days (F1 generation) or 88 days (F1 satellite cross-breeding phase) prior to mating and throughout breeding, gestation and lactation until the recovery period (F1 satellite males only) or necropsy. The F1 satellite males were dosed for at least 114 days prior to the recovery period (33 days). The F1 satellite females were dosed for at least 118 days prior to necropsy.
The offspring of the F1 generation (F2 litters) were potentially exposed to the test article in utero and during lactation days 0-21. The offspring of the F1 satellite animals (F2 satellite litters) were potentially exposed to the test article in utero and during lactation days 0-7.
A dose volume of 5 ml/kg was used in the control, 50, 300 and 1000 mg/kg/day groups. Individual dosages were calculated based on the most recent body weight to provide the correct mg/kg/day dose.

Frequency of treatment:

daily

Remarks:

Doses / Concentrations:
0, 50, 300 and 1000 mg/kg b.wt./day administered in a dose volume of 5 mL/kg b.wt./day
Basis:
actual ingested

No. of animals per sex per dose:

30 rats/sex/dose for all dose levels and control

Control animals:

yes, concurrent vehicle

Positive control:

No data

Parental animals: Observations and examinations:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical observations were recorded weekly for all parental animals throughout the study period.
Animals were also observed for signs of toxicity at the time of dosing (F0 only) and approximately one hour following treatment throughout the dosing period.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual F0, F1 and F1 satellite male body weights were recorded weekly throughout the study and prior to the scheduled necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
Male and female food consumption was measured weekly until pairing. Food intake was not recorded during the mating period. Male food consumption was measured again after mating on a weekly basis until the scheduled necropsy.

Sperm parameters (parental animals):

Gross morphology, an estimate of sperm numbers and the presence or absence of sperm motility were evaluated under a light microscope (100x) by visual estimation. Sperm smears were retained for possible future morphological examination.

Litter observations:

STANDARDISATION OF LITTERS
To reduce variability among the litters, 10 pups per litter, 5 per sex when possible, were randomly selected on lactation day 4. The remaining offspring were weighed, euthanized and discarded following a cursory external examination on lactation day 4.

PARAMETERS EXAMINED
Each litter was examined twice daily for survival and all deaths were recorded. A daily record of litter size was maintained.
Litters were examined daily for survival and any adverse changes in appearance or behavior. Each pup received a detailed physical examination on lactation days 1, 4, 7, 14 and 21.
F1 and F2 pups were individually weighed on lactation days 1, 4, 7, 14 and 21.
Pups were individually sexed on lactation days 0, 4 and 21

GROSS EXAMINATION OF DEAD PUPS:
Intact offspring dying from lactation days 0 to 4 were necropsied

Postmortem examinations (parental animals):

SACRIFICE
Surviving animals were euthanized by carbon dioxide inhalation.
All surviving F0 adults were euthanized following the selection of the F1 generation and completion of a detailed clinical observation. All surviving F1 adults were euthanized following weaning of the F2 pups. All surviving F2 satellite adults were necropsied following completion of the F2 satellite pup necropsies.

GROSS NECROPSY
The necropsy included examination of the external surface, all orifices, the cranial cavity, the external and cut surfaces of the brain and spinal cord, and the thoracic, abdominal and pelvic cavities including viscera. At the time of necropsy the following F0 and F1 parental tissues and organs were collected and were placed in 10% neutral buffered formalin:
Adrenals
Aorta
Bone with marrow (sternebrae)
Brain (forebrain, midbrain, hindbrain)
Coagulating gland
Eyes with optic nerve
Gastrointestinal tract
Esophagus
Stomach
Duodenum
Jejunum
Ileum
Cecum
Colon
Rectum
Heart
Kidneys
Liver (sections of two lobes)
Lungs (including bronchi, fixed by inflation with fixative)
Lymph node (mesenteric)
Ovaries and oviducts
Pancreas
Peripheral nerve (sciatic)
Pituitary
Prostate
Salivary gland [submaxillary]
Seminal vesicles
Skeletal muscle (vastus medialis)
Skin with mammary gland
Spinal cord (cervical)
Spleen
Testes with epididymides and vas deferens
Thymus
Thyroids [with parathyroids, if present]
Trachea
Urinary bladder
Uterus with vagina
All gross lesions

ORGAN WEIGHTS
The testes, epididymides or ovaries and the brain, pituitary gland, kidneys and liver were weighed fresh for all F0 and F1 parental animals euthanized at the scheduled necropsies. Testis, epididyrnis and pituitary gland weights were collected for the F1 satellite males. Absolute weights and organ to final body weights were calculated.

HISTOPATHOLOGY
The tissues indicated in were prepared for microscopic examination:
Cervix
Coagulating gland
Epididymides
Kidneys
Liver
Ovaries
Pituitary gland
Prostate
Seminal vesicles
Testes
Uterus
Vagina
Vas deforens
All internal gross lesions

Postmortem examinations (offspring):

All pups were euthanized with carbon dioxide inhalation. Necropsy examinations of pups emphasized developmental morphology.

Statistics:

Body weight, food consumption, organ weight, litter size, pre-coital and gestation interval data were analyzed by ANOVA with a Dunnett’s
post-hoc test. Pup sex ratio, numbers of stillborn and dead pups, mating, fertility and viability indices were analyzed with the Chi-square test with
Yate’s correction factor. Microscopic pathology data of the control and high-dose groups were compared with the Kolmogorov-Smirnov test.

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

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

not specified

Reproductive function: oestrous cycle:

not specified

Reproductive function: sperm measures:

not specified

Reproductive performance:

effects observed, treatment-related

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
Salivation and yellow, red, brown, clear and/or tan staining/matting/material were noted frequently in the 1000 mg/kg/day group one hour following dosing (males and females).
An increased incidence of red discharge from the vaginal opening was noted for females in the high dose group one hour following dosing, Several occurrences of salivation (females only) and clear, yellow, tan and/or red staining/matting/material around the mouth were noted for animals in the 300 mg/kg/day group.
No clinical signs that could be related to test article administration were observed in the 50 mg/kg/day group males and females.

BODY WEIGHT (PARENTAL ANIMALS)
Mean weekly body weights in the 1000 mg/kg/day group males were reduced and statistically significant (p < 0.01 or p < 0.05) when compared to the control group values beginning week 3 and continuing through week 20 (scheduled necropsy). Mean body weight gains in the 1000 mg/kg/day group males were significantly reduced (p <0.05 or p< 0.01) in comparison to the control group values during weeks 1-2, 7-8, 8-9, 9-10, 11-12, 15-16, 16-17 and 19-20. All other values were similar to the values observed in the control group.
Mean weekly body weights in the 1000 mg/kg/day group females prior to breeding (weeks 0-10) and during weeks 17-20 were similar to the values observed in the control group. Mean body weight gains in the 1000 mg/kg/day group females were significantly reduced (p <0.05 and p <0.01) during weeks 0-1 and 7-8, respectively. All other values were similar to the control group values.
Mean weekly body weights in the 300 mg/kg/day group males were comparable to the control group values during weeks 0-13. Mean body weights in the mid dose group males were significantly reduced (p <0,05) beginning week 14 and continuing through week 20 (scheduled necropsy). Mean body weight gains in the 300 mg/kg/day group males were significantly reduced (p<0.05 or p<0.01) during weeks 3-4, 7-8, 9-10 and 18-19. All other values were similar to the control group values.
Mean weekly body weights in the 300 mg/kg/day group females were comparable to the control group values prior to breeding (weeks 0-10) and during weeks 17-20. Mean body weight gains in the 300 mg/kglday group females were significantly reduced (p< 0.05) during weeks 0-1 and 7-8. All other values were similar to the control group values.
Mean weekly body weights and body weight gains in the 50 mg/kg/day group males and females were not adversely affected by test article administration.

FOOD INTAKE (PARENTAL ANIMALS)
Food consumption, evaluated as g/animal/day and g/kg/day, in the 50, 300 and 1000 mg/kg/day group males and females was unaffected by test article administration throughout the study.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
Reproductive performance was adversely affected by test article administration at a dose level of 1000 mg/kg/day. Fertility indices for the F0 males were 96.7%, 86.7%, 93.3 % and 73.3 % and for the F0 females were 96.7% , 93.3 %, 93,3% and 73.3 % in the control, 50, 300 and 1000 mg/kg/day groups, respectively.

ORGAN WEIGHTS (PARENTAL ANIMALS)
Mean absolute liver weight in the 1000 mg/kg/day group females was significantly increased (p< 0.01) when compared to the control group value (16%). Mean absolute epididymides and ovary weights in the 1000 mg/kg/day group males and females were lower (8% and 19%, respectively) than the control group values; the differences were statistically significant at p< 0.01.
Mean kidney weights were statistically increased (p< 0.01) in the 300 (relative only) and 1000 (absolute and relative) mg/kg/day group males.
No adverse effects on absolute and relative organ weights were noted in the 50 mg/kg/day group males and females.

GROSS PATHOLOGY (PARENTAL ANIMALS)
At the scheduled necropsy, no test article related macroscopic findings were observed in the F0 parental animals. Dark red lungs were noted for two control group males and two, one, two and one females in the control, 50, 300 and 1000 mg/kg/day groups, respectively. Other findings in the treated groups, such as reddened adrenal glands, dilated renal pelvis, pale liver, cystic ovary, accessory spleen, spleens with cyst(s), hemorrhagic thymus gland, clear fluid-filled uterus and reddened lymph nodes, were limited to singular or infrequent occurrences and/or were noted similarly in the control group.

HISTOPATHOLOGY (PARENTAL ANIMALS)
No microscopic lesions attributed to test article administration were observed in any F0 tissues upon histopathological examination. The lesions observed in the treated group males and females were noted infrequently and/or similarly in the control group.

Key result

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Reproductive toxicity Decreased female fertility, prenatal toxicity (increased number of dead pups and reduced litter weight at birth), decreased maternal body weight gain during gestation, and decreased maternal food consumption during lactation.

Clinical signs:

effects observed, treatment-related

Mortality / viability:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Sexual maturation:

not specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

VIABILITY (OFFSPRING)
The number of dead pups on lactation day 0 in the 1000 mg/kg/day group was significantly higher (p< 0.01) than the control group value. The proportion of pups found dead in the 1000 mg/kg/day group was 11.5% (15 dead of 131 total pups born), The numbers of dead pups on lactation day 0 were unaffected by treatment at dose levels of 50 and 300 mg/kg/day. Pup viability indices in the 50, 300 and 1000 mg/kg/day groups were comparable to the control group value on lactation day 1.

CLINICAL SIGNS (OFFSPRING)
The predominant clinical findings were noted for animals in the 1000 mg/kg/day group one hour following dosing and included yellow, brown, tan, clear and/or red staining/matting/material on several body surfaces and salivation.
No clinical signs that could be related to test article administration were observed in the 50 and 300 mg/kg/day group males and females.

BODY WEIGHT (OFFSPRING)
Mean weekly body weights in the 1000 mg/kg/day group males were reduced (statistically significant at p <0.01) when compared to the control group values beginning week 20 and continuing through week 38 (scheduled necropsy).
Mean weekly body weights in the 300 mg/kg/day group males were comparable to the control group values during weeks 20-22.
Mean weekly body weights in the 300 mg/kg/day group females were comparable to the control group values prior to breeding. During weeks 22-23, 23-24 and 24-25, mean body weight gains in the 300 mg/kg/day group females were significantly (p< 0.01) reduced, increased and reduced, respectively.

ORGAN WEIGHTS (OFFSPRING)
Mean absolute liver weight in the 1000 mg/kg/day group females was significantly increased (p<0005) when compared to the control group value (11 %). Mean absolute epididymides, testes and ovary weights in the 1000 mg/kg/day group males and females were lower (17%, 8% and 11%, respectively) than the control group values; the differences for the epididymides and testes were statistically significant at p < 0.01 and p < 0.05, respectively. Increased mean absolute pituitary gland weights were noted for the 1000 mg/kg/day group males and females (26% and 20%, respectively) in comparison to the control group values; the differences were significant at p<0.01.
No adverse effects on absolute and relative organ weights were noted in the 50 mg/kg/day group males and females.

GROSS PATHOLOGY (OFFSPRING)
At the scheduled necropsy, no test article related macroscopic findings were observed in the F1 parental animals.

HISTOPATHOLOGY (OFFSPRING)
No microscopic lesions attributed to test article administration were observed in any F1 tissues upon histopathological examination.

OTHER FINDINGS (OFFSPRING)
Reproductive performance was adversely affected by test article administration at a dose level of 1000 mg/kg/day, Fertility indices for the F1 males were 90.0%, 83.3%. 93.3% and 76.7% and for the F1 females were 93.3%, 93.3%, 100.0% and 76.7% in the control, 50, 300 and 1000 mg/kg/day groups, respectively.
No adverse effects on reproductive performance were noted at dose levels of 50 and 300 mg/kg/day.

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

50 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

organ weights and organ / body weight ratios

gross pathology

Reproductive effects observed:

not specified

Results of the satellite cross-mating phase suggested that these effects upon reproductive performance were attributable to treatment of the female. Neonatal toxicity at the 1000 mg/kg/day dose level was expressed by an increased number of dead pups on lactation day 0 and reduced pup body weights. Equivocal neonatal toxicity was observed at the 300 mg/kg/day dose level by an increased number of dead F1 pups on lactation day 0. This effect was considered to be of equivocal biological significance as it was not reproduced in the F2 pups. No neonatal toxicity was apparent at a dose level of 50 mg/kg/day.

Conclusions:

Based on the results of this study, a dose level of 50 mg/kg/day was considered to be the clear NOAEL (no observable adverse effect level) for systemic parental and neonatal toxicity and 300 mg/kg/day was considered to be the NOAEL for reproductive toxicity. There was no evidence of cumulative toxic effect across generations in this study.

Executive summary:

A two generation reproductive toxicity test was conducted in accordance with GLP to OECD guideline 416. In the study, parental toxicity in the F0 and F1 (including the satellite phase) generations was exhibited at a dose level of 1000 mg/kg/day by mortality (females only), clinical signs, inhibition of body weight gain, increased pituitary gland (F0, Fl and F1 satellite males and F0 and F1 females) and liver (F0 and Fl females) weights and decreased testes (Fl and F1 satellite), epididymides (F0, F1 and F, satellite) and ovary (F0 and F1) weights. Slight parental toxicity was also apparent at the 300 mg/kg/day dose level by clinical signs, reduced body weight gain and increased pituitary gland weights (F0 males only). No parental toxicity was observed at a dose level of 50 mg/kg/day. Reproductive performance was adversely affected (reduced fertility indices, apparent dystocia and reduced live litter size) by test article administration at a dose level of 1000 mg/kg/day.

Results of the satellite cross-mating phase suggested that these effects upon reproductive performance were attributable to treatment of the female. Neonatal toxicity at the 1000 mg/kg/day dose level was expressed by an increased number of dead pups on lactation day 0 and reduced pup body weights. Equivocal neonatal toxicity was observed at the 300 mg/kg/day dose level by an increased number of dead F1 pups on lactation day 0. This effect was considered to be of equivocal biological significance as it was not reproduced in the F2 pups. No neonatal toxicity was apparent at a dose level of 50 mg/kg/day.

Based on the results of this study, a dose level of 50 mg/kg/day was considered to be the clear NOAEL (no observable adverse effect level) for systemic parental and neonatal toxicity and 300 mg/kg/day was considered to be the NOAEL for reproductive toxicity. There was no evidence of cumulative toxic effect across generations in this study.

The study is being used as read across to a structurally similar substance.

 

Effect on fertility: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

150 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Additional information

There are no available data for this endpoint for CAS: 96152-43-1, therefore studies from supporting, structurally similar substances have been used as for read-across purposes to address this endpoint. The NOAEL is then adjusted for the presence of oil in the tested sample (50%) and the lack of this in the Registration material.

Oral

In the key study for reproductive toxicity, oral exposure (Nemec, 1995, report number: WIL-187006) the study was conducted according to OECD Guideline 416 (Two-Generation Reproduction Toxicity Study). The study was conducted in line with GLP.

The reliability rating for this study is 1, however this is being used as read across as there was no available data to fulfil this endpoint for the test material and so the reliability rating will be reduced to 2, according to the criteria of Klimisch, 1997. T

his was considered to be the most reliable and robust study and a full multigenerational study as opposed to a screening study. The NOAEL for reproductive toxicity for this study was 300mg/kg bw/day, which was reduced for the Registration material to 150mg/kg bw/day.

Supporting reproductive screening studies:

- The Lamb, 1993, reproductive toxicity, oral exposure study (report number: WIL-187001) was not considered the key study as it was conducted less recently than the above key study and was considered less robust than the above key study due to its nature as a screening study. This

study was conducted to the OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test) and GLP.

The reliability rating for this study is 1, however this is being used as read across to a supporting substance and so the reliability rating will be reduced to 2, according to the criteria of Klimisch, 1997.

Based upon the results of this study, the apparent NOAEL (no observable adverse effect level) parental toxicity was considered to be 200 mg/kg/day. Equivocal prenatal toxicity was observed at a dose level of 200 mg/kg/day. The results of this screening study indicated that further toxicity testing for adverse reproductive effects should be considered. As such the OECD 416 study (key study) mentioned above was conducted..

- The Schroeder, 1998 study (Huntingdon Life Sciences report number: 96-4084) was

conducted according to OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test). The study was conducted in line with GLP.

The reliability rating for this study is 1, however this is being used as read across to a supporting substance as there was no available data to fulfil this endpoint for the test material and so the reliability rating will be reduced to 2, according to the criteria of Klimisch, 1997. Reproductive toxicity NOAEL = 1000 mg/kg b.wt./day for males and females, highest dose tested.

Short description of key information:
Oral:
Two-generation reproduction study - Based on the results of this study, a NOAEL (no observable adverse effect level) for systemic parental and neonatal toxicity was derived for the test substance. 300 mg/kg/day was also considered to be the NOAEL for reproductive toxicity. There was no evidence of cumulative toxic effect across generations in this study. TO account for the presence of oil in the tested sample (50%), and the lack of oil in the Registration material, the NOAEL was reduced to 150mg/kg bw/day. It is recognised that the oil is not likely to have an effect on reproductive performance and so this reduced NOAEL will be very conservative; the true value for the registration material is more likely to be close to or the same as that of the material in oil. However a precautionary approach has been taken throughout the dossier.

Justification for selection of Effect on fertility via oral route:
Decreased female fertility, prenatal toxicity (increased number of dead pups and reduced litter weight at birth), decreased maternal body weight gain during gestation, and decreased maternal food consumption during lactation. The NOAEL of 300mg/kg bw/day was reduced to account for the presence of oil to 150mg/kg bw /day

Effects on developmental toxicity

Description of key information

Oral:
Maternal toxicity (reduced body weight gain) was observed at the 1000 mg/kg/day dose level. No maternal toxicity was apparent at the 50 and 300 mg/kg/day dose levels. Developmental toxicity was apparent at a dose level of 1000 mg/kg/day by an increased incidence of the skeletal variant bent ribs. No developmental toxicity was observed at dose levels of 50 and 300 mg/kg/day. 
Based on the results of this study, the NOAEL (no observable adverse effect level) for maternal and developmental toxicity was considered to be 300 mg/kg/day. This was then adjusted to account for the presence of oil in the tested samples and the lack in the Registration material, producing a NOAEL for the CSA of 150mg/kg bw/day. 

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

1st to 25th February 1994

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Guideline study following GLP. The reliability is reduced as the study is being used for read-across purposes.

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OTS 798.4900 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc., Portage, Michigan
- Age at study initiation: The animals were approximately eleven weeks old upon receipt.
- Weight at study initiation: The animals were weighed the day following receipt. Body weights ranged from 186 g to 242 g
- Housing: Upon arrival and until pairing, all animals were individually housed in clean, wire-mesh cages suspended above cage-board. The animals were paired for mating in the home cage of the male. Following positive identification of mating, the females were returned to an individual suspended wire-mesh cage.
- Diet (e.g. ad libitum): The basal diet used in this study was Purina® Certified Rodent Chow® #5002. This diet is a certified feed with appropriate analyses performed by the manufacturer and provided to WIL Research laboratories, Inc. No contaminants were present in animal feed or water at levels expected to interfere with the objectives of this study. Basal diet was provided ad libitum throughout the acclimation period and during the study.
- Water (e.g. ad libitum): Municipal water supplying the facility is sampled for contaminants according to Standard Operating Procedures. The results of these analyses are maintained at WIL Research Laboratories, Inc. Drinking water delivered by an automatic watering system.
- Acclimation period: 11 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): The temperature ranged from 67° to 75°F
- Humidity (%): relative humidity ranged from 20% to 49 %
- Air changes (per hr): Air handling units were set to provide approximately 10 fresh air changes per hour.
- Photoperiod (hrs dark / hrs light): 12-hour light/12-hour dark

Route of administration:

oral: gavage

Vehicle:

peanut oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
An appropriate amount of the test material was drawn into a syringe for each group. A sufficient amount of the vehicle, peanut oil, was added to a properly-labeled storage container. The test material was slowly added to the vehicle while being mixed on a Polytron® PT6000 until all of the test material was incorporated into the vehicle. The mixtures were stirred using a magnetic stir bar and plate throughout sampling, dispensation, dosing and periods of non-use.
Preparations for all dose groups were formulated three times (January 11 and February 7 and 14, 1994). The preparations were stored at room temperature, protected from light. The formulated suspensions were visually inspected for homogeneity by the Study Director prior to the initiation of dosing and were found to be acceptable for administration.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dosing solutions in peanut oil vehicle were prepared three times over a period of about a month, and their concentration, homogeneity and stability were verified by chemical analysis.

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused: The animals were paired for mating in the home cage of the male.
- M/F ratio per cage: The animals were paired on a 1:1 basis.
- Proof of pregnancy: Positive evidence of mating was confirmed by the presence of a copulatory plug or the presence of sperm in a vaginal smear. Each mating pair was examined daily. The day on which evidence of mating was identified was termed day 0 of gestation and the animals were separated.

Duration of treatment / exposure:

Exposure period was gestation days 6 through 15. For the control, 25 mated females received peanut oil vehicle only.
A dosage volume of 5 ml/kg was used for all dosage levels.

Frequency of treatment:

daily

Duration of test:

Dams euthanized for uterine examinations and collection of fetuses on gestation day 20.

No. of animals per sex per dose:

25 females per dose

Control animals:

yes, concurrent vehicle

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All rats were observed twice daily for moribundity and mortality. Animals were also observed for signs of toxicity approximately one hour following treatment throughout the dosing period.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical observations were recorded individually from days 0 through 20 of gestation.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual maternal body weights were recorded on gestation days 0, 6-16 (daily), and 20.

FOOD CONSUMPTION: Yes
Individual food consumption was recorded on gestation days 0, 6-16 (daily) and 20. Food intake was reported as g/animal/day and g/kg/day for each corresponding body weight change interval.

POST-MORTEM EXAMINATIONS: Yes
- All maternal animals were euthanized by carbon dioxide inhalation on gestation day 20.
- Organs examined:
The thoracic, abdominal and pelvic cavities were opened by a ventral mid-line incision and the contents were examined. In all instances, the post mortem fmdings were correlated with the ante mortem comments and any abnormalities were recorded.

Ovaries and uterine content:

The uterus and ovaries were excised and the number of corpora lutea on each ovary was recorded. The trimmed uterus was weighed, opened and the number and location of all fetuses, early and late resorptions and the total number of implantation sites were recorded. The individual uterine distribution of implantation sites was documented using the following procedure. All implantation sites, including resorptions, were numbered in consecutive order beginning with the left distal to the left proximal uterine horn, noting the position of the cervix, and continuing from the right proximal to the right distal uterine horn. Maternal tissues were preserved in 10% neutral buffered formalin for possible future histopathological examination only as indicated by the gross findings. The carcass of each dam was then discarded.

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: Yes: half per litter

Statistics:

Body and uterine weights, food consumption, corpora lutea, total implantations and viable fetuses were analyzed by ANOVA with a Dunnett’s
post-hoc test. Fetal sex ratios were analyzed with the Chi-square test with Yate’s correction factor. Resorptions, post-implantation losses, and dead fetuses were analyzed with the Mann-Whitney U-test. Litter proportions of intrauterine data were analyzed with the Kruskal-Wallis test. Fetal
malformations and variations were analyzed with Fisher’s Exact test. Mean litter proportions of malformations and variations were analyzed with the Mann-Whitney U-test.

Historical control data:

Yes

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
Decreased mean body weight gain during the dosing period (from gestation days 6 to 10-16) at 1000 mg/kg b.wt./day. No maternal body weight
effects at 300 mg/kg b.wt./day

Key result

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Key result

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
The number of litters with fetuses having skeletal variation (bent ribs) was increased at 1000 mg/kg b.wt./day. This and other fetal variations were
not increased at 300 mg/kg b.wt./day. Therefore the developmental toxicity NOEL was 300 mg/kg/bw/day.

Key result

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

skeletal malformations

Abnormalities:

not specified

Developmental effects observed:

not specified

Statistical results:

Mean body weight gains during the dosing period (from gestation days 6 to 10-16) of the high-dose group were significantly (p<0.01) lower than controls. The number of high-dose litters with fetuses having bent ribs was significantly higher (p<0.05) than the control group. All other statistically significant differences were not of toxicological significance.

Result observations:

Mating success was variable among groups. The number of gravid females/group was 25, 22, 24 and 21 for control, low-, middle- and high-dose groups, respectively. There were no abortions.

Survival: All dams survived to scheduled termination.

Clinical observations: Material and/or staining around the mouth of about half of the high dose animals were the only treatment-related signs and were seen only at the 1-hour post-dose observation time. Onset of these signs usually occurred within 5-7 days after initiation of dosing and signs persisted until the end of the exposure period.

Body weights: There were sporadic and transient differences in mean body weights between treatment groups and controls, due to variation in the number of gravid females/group and differences in day 0 means. Body weight gain, however, in the high-dose group was about 17% lower than controls only during the exposure period, starting on day 10 and persisting until day 16.

Food consumption: There was no effect on food consumption on a g/kg b.wt./day basis.

Gravid uterine weights: There were no significant differences of mean gravid uterine weights.

Maternal gross pathology: No dose-related gross pathology was observed.

Ovarian/intrauterine observations: Embryonic and fetal growth, development and survival were not affected by test material administration. Pre- and post-implantation loss, early and late resorptions, viable and dead fetuses were not affected by treatment when evaluated as litter means or as mean litter proportions. Likewise, sex ratio and weight of fetuses were not affected by treatment. Mean number of viable fetuses ranged from 15.9-17.3 per litter.

Fetal malformations: Malformations were found in 3 fetuses from different litters. These consisted of 2 fetuses from the mid-dose group, one with exencephaly, malpositioned testes and costal cartilage anomaly and the other with hydrocephaly. The third malformed fetus was from the high-dose group and had a single malformation, meningocele. The occurrence and incidence of these malformations was within the range of laboratory historical controls. These malformations are, therefore, considered to be of spontaneous origin.

Fetal variations: The number of litters in the high-dose group with fetuses having bent ribs was significantly higher than the control group. This skeletal variation was not found with elevated frequency in the low- or mid-dose groups. No other treatment related skeletal variations were observed.

Conclusions:

Based on the results of this study, the NOAEL (no observable adverse effect level) for maternal and developmental toxicity was considered to be 300 mg/kg/day.

Executive summary:

The potential maternal and developmental toxicity of the test material was evaluated in the rat by a study conducted to OECD Guideline 414 conducted in accordance with GLP.

The potential maternal and developmental toxicity of the test material was evaluated in the rat. The test material in peanut oil was administered orally by gavage to three groups of 25 bred Sprague-Dawley Crl:CD®BR female rats once daily from gestation days 6 through 15. Dosage levels were 50, 300 and 1000 mg/kg/day administered at a dose volume of 5 m1/kg. A concurrent control group (25 bred females) received the vehicle, peanut oil, on a comparable regimen at 5 ml/kg. All rats were observed twice daily for appearance and behavior. Body weights and food consumption were recorded at appropriate intervals. A laparohysterectomy was performed on all animals on gestation day 20. The uteri and ovaries were examined and the numbers of fetuses, early and late resorptions, total implantations and corpora lutea were recorded. Mean gravid uterine weights and net body weight changes were calculated for each group. The fetuses were weighed, sexed and examined for external, soft tissue and skeletal malformations and developmental variations.

All maternal animals survived to the scheduled necropsy on gestation day 20. Treatment-related clinical signs were noted one hour following dosing in the 1000 mg/kg/day group and consisted of red, clear, yellow and/or tan staining/matting/material around the nose and mouth. Mean body weight gains in the 1000 mg/kg/day group were significantly reduced for gestation days 6-10, 6-11, 6-12, 6-13, 6-14, 6-15 and 6-16. Mean food consumption in the 50, 300 and 1000 mg/kg/day groups was unaffected by test article administration. No treatment-related necropsy fmdings were noted at any dose level on gestation day 20.

Intrauterine growth and survival were unaffected by treatment at dose levels of 50, 300 and 1000 mg/kg/day. The malformations observed in this study were considered to be spontaneous in origin. A significantly increased incidence of one fetal developmental variant, bent ribs, was observed at the 1000 mg/kg/day dose level.

In conclusion, maternal toxicity (reduced body weight gain) was observed at the 1000 mg/kg/day dose level. No maternal toxicity was apparent at the 50 and 300 mg/kg/day dose levels. Developmental toxicity was apparent at a dose level of 1000 mg/kg/day by an increased incidence of the skeletal variant bent ribs. No developmental toxicity was observed at dose levels of 50 and 300 mg/kg/day. Based on the results of this study, the NOAEL (no observable adverse effect level) for maternal and developmental toxicity was considered to be 300 mg/kg/day.

The study is being used as read across to a structurally similar substance.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

150 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Additional information

There are no available data for this endpoint for CAS: 96152-43-1, therefore studies from supporting, structurally similar substances have been used for read-across purposes to address this endpoint. The NOAEL has been adjusted for the presence of oil.

Oral

- In the key study for Developmental toxicity, oral exposure (Nemec, 1994, report number: WIL-187005) the study was conducted according to OECD Guideline 414 (Prenatal Developmental Toxicity Study). The study was conducted in line with GLP.

The reliability rating for this study is 1, however this is being used as read across as there was no available data to fulfil this endpoint for the test material and so the reliability rating will be reduced to 2, according to the criteria of Klimisch, 1997.

Supporting developmental study:

- The Watson, 1990, developmental toxicity, oral exposure study (report number: CEHC 3086) was not considered the key study as it was conducted less recently than the above key study and had significant deviations from the guideline. The study was conducted according to OECD Guideline 414 (Prenatal Developmental Toxicity Study) and GLP, however fewer than 20 gravid females per group and no laboratory historical control data was presented. A reliability rating of 2 according to the criteria of Klimisch, 1997 was assigned. The study is being used as read across to a structurally similar substance. Based upon the results of this study, the maternal no-observed-effect level (NOEL) was 300 mg/kg/day, and the developmental NOEL was 50 mg/kg/day. The developmental effects were not reflected in the key study at this dose level.

Justification for selection of Effect on developmental toxicity: via oral route:
Maternal toxicity (reduced body weight gain) was observed at the 1000 mg/kg/day dose level. No maternal toxicity was apparent at the 50 and 300 mg/kg/day dose levels. Developmental toxicity was apparent at a dose level of 1000 mg/kg/day by an increased incidence of the skeletal variant bent ribs. No developmental toxicity was observed at dose levels of 50 and 300 mg/kg/day. The NOAEL of 300mg/kg bw/day was reduced to account for the presence of oil to 150mg/kg bw /day
Based on the results of this study, the NOAEL (no observable adverse effect level) for maternal and developmental toxicity was considered to be 300 mg/kg/day. These values are then adjusted to account for the presence of oil (50%) in the tested samples to derive a NOAEL for the Registration material of 150mg/kg bw/day for developmental (and maternal) toxicity.

Justification for classification or non-classification

The results from the key study performed on a similar material within the Category which is being used as read-across to this Registration material, show effects on reproductive performance. These are considered most likely to be caused by the presence of a component of the Registration material which is classified as a Category 2 (DSD) or Category 1B (CLP) Reproductive toxin which is present at low levels in the Registration material. Hence the Registration material classification for this endpoint takes this into account and is classified accordingly (see Section 2).