Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

Certain alkyl phenate sulfides such as EC 272-234-3 contain residual amounts of an alkylphenol, TPP (EC 310-154-3). TPP is classified as a category 1B reproductive hazard and is the source of the reproductive toxicity in alkyl phenate sulfides. This is demonstrated by supporting study 1, which tested an alkyl phenate sulfide that was depleted of residual TPP and resulted in no observations of reproductive toxicity in an OECD 415 one-generation study. In addition, a comparison of two OECD 416 two-generation reproductive toxicity studies – one with an alkyl phenate sulfide (EC 272-234-3) and the second with TPP – further demonstrates that it is the TPP causing the reproductive toxicity. Specifically, the residual amount of TPP in EC 272-234-3 is approximately 6.7% and comparing the TPP “equivalent” dose, or dose delivered of TPP in the EC 272-234-3 study yields comparable potency:

·        NOAEL: 20.1 mg/kg/day residual TPP equivalent dose in the EC 272-234-3 study compared to a NOAEL of 15 mg/kg/day in the TPP study

·        LOAEL: 67 mg/kg/day residual TPP equivalent dose in the EC 272-234-3 study compared to a LOAEL of 75 mg/kg/day in the TPP study

A two-generation reproductive toxicity study with TPP (Edwards, 2012) is included as supporting study 2 as this is the key study to address the hazards of TPP. In addition, the risk assessment is conducted on TPP as a surrogate for EC 272-234-3 as TPP is the source of the reproductive toxicity and this study is used to calculate the DNEL.

 

Effects on fertility

Oral:

Key Study 1:

- The reproductive toxicity study 1(Lamb, 1993, report number: WIL-187001) was conducted to the OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test) and GLP. A reliability rating of 1 was assigned 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.

Key Study 2:

The study for reproductive toxicity, oral exposure (Nemec, 1995, report number: WIL-187006) was conducted according to OECD Guideline 416 (Two-Generation Reproduction Toxicity Study), and in line with GLP. A reliability rating of 1 was assigned according to the criteria of Klimisch, 1997. This was considered a reliable and robust study. 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 correspondingly, 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.

Supporting Study 1:

Reproductive toxicity observed with the substance,Phenol, dodecyl-, sulfurized, carbonates, calcium salts, overbased, is attributed to the presence of the impurityPhenol, dodecyl-branched (AKA, tetrapropenyl phenol; EC 310-154-3; CAS 121158-58-5; referred to as “TPP”). Therefore, this supporting study is provided to demonstrate that the residual TPPis thesole source of the reproductive toxicity observed and the substance itself phenol, dodecyl-, sulfurized, carbonates, calcium salts, overbased,does not cause reproductive toxicity.

Alkyl phenate sulfide oligomers were synthesized specifically for testing the reproductive toxicity of thesubstancePhenol, dodecyl-, sulfurized, carbonates, calcium salts, overbasedwith reduced concentrations of TPP. The material was stripped of free TPP so that TPP was present at < 0.2 wt%. While there was no calcium carbonate overbasing in the alkyl phenate sulfide, the remainder of the substance, including the amount of diluent oil present, was analogous to that of EC 272-234-3. The amount of calcium carbonate overbasing is not expected to alter the toxicological properties of the alkyl phenate sulfides and calcium carbonate itself is not a reproductive or developmental hazard.  

 

The TPP-depleted alkyl phenate sulfide oligomers were tested in an OECD 415 one-generation reproductive toxicity study (Edwards, 2012).The study was GLP with a reliability rating of 1 according to the criteria of Klimisch, 1997. This was considered a reliable and robust study.The study resulted in a NOAEL of 1000 mg/kg/day (top dose) for systemic, reproductive, and neonatal toxicity.

 

Supporting Study 2:

 

Parental toxicity was evidenced by lower mean body weights, body weight gains, and food consumption in the F0 and F1 males and females in the 75 mg/kg/day group. In addition, several organ weight changes (lower weights of the cauda epididymides, epididymides, prostate, and seminal vesicles/coagulating glands, and higher pituitary weight for F0 and F1 males; lower left and right testes weight for F1 males; lower ovary weights for F0 and F1 females; and higher adrenal glands weight for F1 females) were noted for parental animals at 75 mg/kg/day. Furthermore, histopathologic changes of renal mineralization in F0 males at 75 mg/kg/day and F1 males at 15 and 75 mg/kg/day, as well as decreased corpora lutea in F0 and F1 females at 75 mg/kg/day were noted. Therefore, the no-observed-adverse-effect level (NOAEL) for F0 and F1 parental toxicity was considered to be 15 and 1.5 mg/kg/day, respectively.

 

Decreased implantation sites (F0 females), increased estrous cycle lengths (F0 and F1 females), and a reduction in mean epididymal sperm concentration (F0 males) were noted at 75 mg/kg/day. Therefore, the NOAEL for male and female reproductive toxicity was considered to be 15 mg/kg/day.

 

Based on reductions in F2 and F2a postnatal survival, lower F1, F2, and F2a offspring body weights and body weight gains (that resulted in a delay in the mean age of balanopreputial separation, lower spleen and thymus weights, and post-weaning mortality) and the accelerated onset of vaginal patency in F1 females at 75 mg/kg/day, the NOAEL for neonatal toxicity was considered to 15 mg/kg/day.

 

The following information is taken into account for any hazard / risk assessment:

EC 272-234-3:

Oral:

Nemec, 1995 (Two-generation reproductive toxicity study - Based on the results of this study, a dose level of 300 mg/kg/ bw/day was considered to be theclearNOAEL (no observable adverse effect level) for reproductive toxicity (effects on fertility).

Value used for CSA (route: oral):NOAEL: 300 mg/kg bw/day

EC 310-154-3 (TPP):

Oral:

Edwards, 2012 (Two-generation reproductive toxicity study -Based on the results of this study, a dose level of 15 mg/kg/ bw/day was considered to be theclearNOAEL (no observable adverse effect level) for reproductive toxicity (effects on fertility). A NOAEL of 1.5 mg/kg bw/day was the NOAEL for parental toxicity based on renal mineralization that occurred in F1 males; note that this is not a reproductive effect but is included here as this was observed in the two-generation reproductive toxicity study.

Developmental toxicity

Oral:

The key reproductive toxicity study (Nemec, 1995) is also relevant for developmental toxicity as this study resulted in neonatal effects.

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. A reliability rating of 1 according to the criteria of Klimisch, 1997. This was considered to be the most reliable study.

Based upon the results of this study, the maternal no-observed-adverse-effect level (NOAEL) was 300 mg/kg/day, and the developmental NOAEL was 50 mg/kg/day.

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 following information is taken into account for any hazard / risk assessment:

Oral:

Nemec, 1995 (Two-generation reproductive toxicity study - Based on the results of this study, a dose level of 50 mg/kg/day was considered to be theclearNOAEL (no observable adverse effect level) for developmental toxicity (neonatal toxicity). The NOAEL was based on an increased number of dead F1 pups on lactation day 0 in the 300 mg/kg bw/day dose group. This effect was not reproduced in the F2 pups but occurred in both the F1 and F2 generations at the 1000 mg/kg bw/day dose group.

Value used for CSA (route: oral):NOAEL: 50 mg/kg bw/day

Justification for classification or non classification

Using the available data the substance is determined to be Reproductive category 2 according to Directive 67/548/EEC and is labelled as Repro. Cat. 2; R60: May impair fertility.

In Regulation (EC) no 1272/2008, the test substance is considered to be classified as Repro Category 1B; H360: May damage fertility or the unborn child <fertility> <oral>.

Remarks: Classification represents substance as manufactured containing the impurity tetrapropenyl phenol. This impurity contributes to the hazards of the substance resulting in classification for reproductive effects.

Link to relevant study records
Reference
Endpoint:
two-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18th March 1994 to 17th February 1995 (experimental phase)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study following GLP.
Qualifier:
according to
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 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:
F0 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:
Not applicable
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.
Increased mean absolute pituitary gland weights were noted for the 300 mg/kg bw/day group males (15%) and the 1000 mg/kg bw/day males and females (22% & 9%, respectively) in comparison to the control group values; the differences for the males were 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.

Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
body weight and weight gain
organ weights and organ / body weight ratios
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive performance
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.
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
50 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
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.

Effect on fertility: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
300 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
A reliability rating of 1 was assigned according to the criteria of Klimisch, 1997.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

Effects on fertility

Oral:

Key Study 1:

- The reproductive toxicity study 1(Lamb, 1993, report number: WIL-187001) was conducted to the OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test) and GLP. A reliability rating of 1 was assigned 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.

Key Study 2:

The study for reproductive toxicity, oral exposure (Nemec, 1995, report number: WIL-187006) was conducted according to OECD Guideline 416 (Two-Generation Reproduction Toxicity Study), and in line with GLP. A reliability rating of 1 was assigned according to the criteria of Klimisch, 1997. This was considered a reliable and robust study. 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 correspondingly, 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.

Supporting Study 1:

Reproductive toxicity observed with the substance,Phenol, dodecyl-, sulfurized, carbonates, calcium salts, overbased, is attributed to the presence of the impurityPhenol, dodecyl-branched (AKA, tetrapropenyl phenol; EC 310-154-3; CAS 121158-58-5; referred to as “TPP”). Therefore, this supporting study is provided to demonstrate that the residual TPPis thesole source of the reproductive toxicity observed and the substance phenol, dodecyl-, sulfurized, carbonates, calcium salts, overbased,does not cause reproductive toxicity.

Alkyl phenate sulfide oligomers were synthesized specifically for testing the reproductive toxicity of thesubstancePhenol, dodecyl-, sulfurized, carbonates, calcium salts, overbasedwith reduced concentrations of TPP. The material was stripped of free TPP so that TPP was present at < 0.2 wt%. While there was no calcium carbonate overbasing in the alkyl phenate sulfide, the remainder of the substance, including the amount of diluent oil present, was analogous to that of EC 272-234-3. The amount of calcium carbonate overbasing is not expected to alter the toxicological properties of the alkyl phenate sulfides and calcium carbonate itself is not a reproductive or developmental hazard.  

 

The TPP-depleted alkyl phenate sulfide oligomers were tested in an OECD 415 one-generation reproductive toxicity study (Edwards, 2012).The study was GLP with a reliability rating of 1 according to the criteria of Klimisch, 1997. This was considered a reliable and robust study.The study resulted in a NOAEL of 1000 mg/kg/day (top dose) for systemic, reproductive, and neonatal toxicity.

 

Supporting Study 2:

 

Parental toxicity was evidenced by lower mean body weights, body weight gains, and food consumption in the F0 and F1 males and females in the 75 mg/kg/day group. In addition, several organ weight changes (lower weights of the cauda epididymides, epididymides, prostate, and seminal vesicles/coagulating glands, and higher pituitary weight for F0 and F1 males; lower left and right testes weight for F1 males; lower ovary weights for F0 and F1 females; and higher adrenal glands weight for F1 females) were noted for parental animals at 75 mg/kg/day. Furthermore, histopathologic changes of renal mineralization in F0 males at 75 mg/kg/day and F1 males at 15 and 75 mg/kg/day, as well as decreased corpora lutea in F0 and F1 females at 75 mg/kg/day were noted. Therefore, the no-observed-adverse-effect level (NOAEL) for F0 and F1 parental toxicity was considered to be 15 and 1.5 mg/kg/day, respectively.

 

Decreased implantation sites (F0 females), increased estrous cycle lengths (F0 and F1 females), and a reduction in mean epididymal sperm concentration (F0 males) were noted at 75 mg/kg/day. Therefore, the NOAEL for male and female reproductive toxicity was considered to be 15 mg/kg/day.

 

Based on reductions in F2 and F2a postnatal survival, lower F1, F2, and F2a offspring body weights and body weight gains (that resulted in a delay in the mean age of balanopreputial separation, lower spleen and thymus weights, and post-weaning mortality) and the accelerated onset of vaginal patency in F1 females at 75 mg/kg/day, the NOAEL for neonatal toxicity was considered to 15 mg/kg/day.

The following information is taken into account for any hazard / risk assessment:

EC 272-234-3:

Oral:

Nemec, 1995 (Two-generation reproductive toxicity study - Based on the results of this study, a dose level of 300 mg/kg/ bw/day was considered to be theclearNOAEL (no observable adverse effect level) for reproductive toxicity (effects on fertility).

Value used for CSA (route: oral):NOAEL: 300 mg/kg bw/day

EC 310-154-3 (TPP):

Oral:

Edwards, 2012 (Two-generation reproductive toxicity study -Based on the results of this study, a dose level of 15 mg/kg/ bw/day was considered to be theclearNOAEL (no observable adverse effect level) for reproductive toxicity (effects on fertility). A NOAEL of 1.5 mg/kg bw/day was the NOAEL for parental toxicity based on renal mineralization that occurred in F1 males; note that this is not a reproductive effect but is included here as this was observed in the two-generation reproductive toxicity study.

Effects on developmental toxicity

Description of key information
Nemec, 1995 (Two-generation reproductive toxicity study - 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 developmental toxicity (neonatal toxicity). The NOAEL was based on an increased number of dead F1 pups on lactation day 0 in the 300 mg/kg bw/day dose group. This effect was not reproduced in the F2 pups but occurred in both the F1 and F2 generations at the 1000 mg/kg bw/day dose group. 
Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18th March 1994 to 17th February 1995 (experimental phase)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study following GLP.
Qualifier:
according to
Guideline:
other: OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on test animals 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.
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.
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.
Duration of treatment / exposure:
F0 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
No. of animals per sex per dose:
30 rats/sex/dose for all dose levels and control
Control animals:
yes, concurrent vehicle
Maternal 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.
Fetal examinations:
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
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.
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
NOAEL (parental toxicity): 50 mg/kg bw/day (acual dose received) (male/female)
Based on: Clinical signs, reduced body weight, increased pituitary gland weight (F0 males only at 300 mg/kg bw/day)

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.
Increased mean absolute pituitary gland weights were noted for the 300 mg/kg bw/day group males (15%) and the 1000 mg/kg bw/day males and females (22% & 9%, respectively) in comparison to the control group values; the differences for the males were 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.
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
Details on embryotoxic / teratogenic effects:
Details on embryotoxic / teratogenic effects:
NOAEL (neonatal toxicity): 50 mg/kg bw/day (actual dose received) (male/femal)
Based on: Increased number of dead pups at on lactation day 0 (F1 pups only at 300 mg/kg bw/day)

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.
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
changes in postnatal survival
Abnormalities:
not specified
Developmental 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.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
50 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
A reliability rating of 1 was assigned according to the criteria of Klimisch, 1997.
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

Oral:

The key reproductive toxicity study (Nemec, 1995) is also relevant for developmental toxicity as this study resulted in neonatal effects.

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. A reliability rating of 1 according to the criteria of Klimisch, 1997. This was considered to be the most reliable study.

Based upon the results of this study, the maternal no-observed-adverse-effect level (NOAEL) was 300 mg/kg/day, and the developmental NOAEL was 50 mg/kg/day.

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 following information is taken into account for any hazard / risk assessment:

Oral:

Nemec, 1995 (Two-generation reproductive toxicity study - Based on the results of this study, a dose level of 50 mg/kg/day was considered to be theclearNOAEL (no observable adverse effect level) for developmental toxicity (neonatal toxicity). The NOAEL was based on an increased number of dead F1 pups on lactation day 0 in the 300 mg/kg bw/day dose group. This effect was not reproduced in the F2 pups but occurred in both the F1 and F2 generations at the 1000 mg/kg bw/day dose group.


Justification for selection of Effect on developmental toxicity: via oral route:
The key reproductive toxicity study (Nemec, 1995) is also relevant for developmental toxicity as this study resulted in neonatal effects. This study was conducted according to OECD Guideline 416 (Two-Generation Reproduction Toxicity Study), and in line with GLP.

Justification for classification or non-classification

Using the available data the substance is determined to be Reproductive category 2 according to Directive 67/548/EEC and is labelled as Repro. Cat. 2; R60: May impair fertility.

In Regulation (EC) no 1272/2008, the test substance is considered to be classified as Repro Category 1B; H360: May damage fertility or the unborn child <fertility> <oral>.

Remarks: Classification represents substance as manufactured containing the impurity phenol, dodecyl-, branched. This impurity contributes to the hazards of the substance resulting in classification for reproductive effects.