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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
two-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
12 July 2004 - 2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed according to OECD guideline

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2006

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3800 (Reproduction and Fertility Effects)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
4-tert-butylphenol
EC Number:
202-679-0
EC Name:
4-tert-butylphenol
Cas Number:
98-54-4
Molecular formula:
C10H14O
IUPAC Name:
4-tert-butylphenol
Details on test material:
- Name of test material (as cited in study report): para-tertiary butylphenol, UCAR Butylphenol 4T-Flake (para tert-Butyl Phenol), PTBP.
- Physical state: White flakes.
- Analytical purity: Not reported.
- Lot/batch No.: Charge No. 496500; BRRC Sample No. 48-186.
- Expiration date of the lot/batch: Not reported.
- Stability under test conditions: Not reported.
- Storage condition of test material: Not reported.

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River UK Ltd., Margate, Kent, UK
- Age at study initiation: On arrival the animals were ca 4 weeks of age
- Weight at study initiation: On arrival: 93-123 g for males and 95-141 g for females

- Housing: F0 animals were initially housed 2 per cage in polypropylene cages with stainless steel grid bottoms and mesh tops and measured ca 58 x 38.5 x 20 cm. Male and female cages were racked separately. Three days prior to mating, the males were transferred to individual grid bottomed
cages. For mating, the females were transferred to the cage of the appropriate co-group male. Mated females were transferred to individual
solid-bottomed cages, measuring ca 42 x 27 x 20 cm, in which sterilised white wood shavings were provided as bedding. Just prior to anticipated
parturition, some white paper tissue was provided as nesting material. Females retained this type of caging until termination. F1 animals retained
after weaning were housed 2 per cage in ca 42 x 27 x 20 cm grid bottomed cages, sexes separate. At a convenient time, the animals were transferred to larger cages as previously described. Three days prior to mating, the F1 males were transferred to individual grid bottomed cages. The F1 animals then followed the same caging regime as described for the F0 animals. Cages, absorbent papers and water bottles were changed at regular intervals, as appropriate. Nesting material was changed when it became unacceptably soiled and was withdrawn at a suitable time during lactation. Clean wood
shavings were provided at each change of solid-bottomed cages.

- Diet: Ad libitum - Breeder Diet No. 3 (Expanded) SQC (ground), was supplied by Special Diets Services Limited, Stepfield, Witham, Essex, UK
- Water: Ad libitum
- Acclimation period: Charles River Laboratories animal room for 13 days prior to commencement of treatment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Raget 20°C ± 2°C. Actual range 16-21°C.
- Humidity (%): Target 50% ± 15%. Actual actual range was 24-76%.
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): Light hours were 0700-1900 hours

Administration / exposure

Route of administration:
oral: feed
Vehicle:
other: Diet
Details on exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): Fresh diets were prepared as required but no less frequently than weekly during the
study. Each mixed batch was stored in a closed container at ambient temperature and used within the 10 day stability period.
The diet contained a constant concentration of test item and was available continuously to the animals ad libitum.

- Mixing appropriate amounts with (Type of food): An appropriate weight of premix, at a concentration of 100000 p.p.m., was prepared by
adding the required weight of test item to the corresponding weight of untreated diet. Acetone was used to incorporate the test item into the premix; the incorporation rate was 100 mL of solvent per kg premix. The premix was mixed for ca 1h on a Hobart planetary mixer with fan assisted venting,
to aid removal of acetone. A control premix was prepared using acetone and blank diet in the same proportion.
Concentrations of the test substance in the final diet mix were 0, 800, 2500 and 7500 ppm.
- Storage temperature of food: Ambient Room temp

Details on mating procedure:
Over the 2 weeks prior to pairing for mating the F0 and F1 animals, vaginal lavages were taken early each morning and the stage of oestrus observed was recorded.

- M/F ratio per cage: Animals were paired on a one male to one female basis, siblings and other closely related pairings being avoided.
- Length of cohabitation: 14 days
- Proof of pregnancy: The day of detection of a copulatory plug in situ and/or of sperm in the lavage was designated Day 0 of gestation.
- At the end of the mating period, if no mating sign was observed, attempts to breed from the female was ceased and she was returned to an
individual, solid bottomed cage.
- Following completion of mating, the males were returned to group housing with their original cage-mate until termination.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of diet were analysed on 6 occasions, but in response to some values being outside ± 10% of the nominal, additional samples were
taken to investigate the cause of the divergence. As a result formulated diets were sampled on 10 occasions (one set taken as contingency) and
analysed on 9 occasions during the study treatment period, and the premix on 2 occasions. The procedures for additional sampling were standard
procedures and considered best practice; the deviation from the protocol did not affect the integrity of the study. On each occasion, triplicate
samples were withdrawn from each formulation containing test item, and the Control diet.

- Formulated diets prepared for the first week of the study were within the acceptance criteria at 800 and 7500 p.p.m. but slightly outwith the
acceptance criteria of ± 10% (10.6%) at 2500 p.p.m.
- From diets prepared for use on Week 8 of the study, the diet at 7500 p.p.m. was within the acceptance criteria, both replicates at 800 p.p.m. were outwith the acceptance criteria, and at 2500 p.p.m. one of the replicates was outwith the acceptance criteria. The largest difference from nominal
was -13.1%.
- All of the samples prepared for use on Week 14 and 20 of the study were within ± 7% of the nominal dietary concentration.
- During Week 28 the analysed concentration of the samples was 22.3%, 23.0% and 16.4% lower than the nominal at 800, 2500 and 7500 p.p.m,
respectively.
- An additional sampling occasion was performed during Week 30; the analysed concentration of the samples was 15.9%, 18.9% and 15.2% lower than
the nominal at 800, 2500 and 7500 p.p.m., respectively.
- During Week 35 the analysed concentration of the samples was 22.1%, 27.1% and 22.2% lower than the nominal at 800, 2500 and 7500 p.p.m.,
respectively.
- An additional sampling occasion was performed during Week 36; the analysed concentration of the samples was 29.3%, 27.6% and 22.8% lower than
the nominal at 800, 2500 and 7500 p.p.m., respectively.
- A second additional sampling occasion was performed during Week 37; the analysed concentration of the samples was 11.0%, 9.6% and 7.2% lower
than the nominal at 800, 2500 and 7500 p.p.m., respectively. The premix concentration was analysed and the concentration was 0.8% different from the nominal concentration.
The analysis of the Control diet revealed low level contamination on three occasions. At these low levels none of these occurrences was considered
to have affected the integrity of the study.
Duration of treatment / exposure:
F0 animals were treated for 10 weeks prior to mating, commencing at ca 6 weeks of age. Treatment continued for both sexes throughout mating, gestation and lactation until termination.
The selected F1 animals were treated for at least 10 weeks after weaning, prior to mating. Treatment was then continued for both sexes
throughout mating, gestation and lactation until termination of the F1 adults and F2 weanlings.
Frequency of treatment:
Ad libitum in diet
Details on study schedule:
- F1 parental animals not mated until 10 weeks after selected from the F1 litters.
- Selection of parents from F1 generation when pups were 21 days of age.
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 70, 200, 600 mg/kg/day
Basis:
nominal in diet
No. of animals per sex per dose:
F0 generation 28 rats/sex/group
F1 generation 24/sex/group
Control animals:
yes, plain diet
Details on study design:
Animals were selected at random
Positive control:
None

Examinations

Parental animals: Observations and examinations:
All animals were checked early morning and as late as possible each day for viability.

DETAILED CLINICAL OBSERVATIONS: Once each week all animals received a detailed clinical examination, which included appearance, movement
and behaviour patterns, skin and hair condition, eyes and mucous membranes, respiration and excreta. All of the animals were examined for
reaction to treatment during the day.

BODY WEIGHT: Weights of F0 animals were recorded one week prior to the first day of test item administration, then weekly thereafter until the start of the mating period. Males then continued weekly weighings until termination, while for females, weighing resumed on Day 0 of gestation (the day of detection of a positive mating sign), followed by Days 7, 14 and 20 of gestation, Days 1, 7, 14 and 21 of lactation (where the day of birth of the
litter is designated Day 0 of lactation). Post-weaning F1 animals were weighed weekly, starting on a suitable day within one week of weaning of the
majority of the litters, until termination for males and until mating commenced for females. Mated F1 females were weighed on Days 0, 7,
14 and 20 of gestation, then on Days 1, 7, 14 and 21 of lactation.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
Both sexes: Weekly, starting one week before treatment began (F0 animals), or from a suitable day within one week of weaning of the majority of the animals (F1 animals), until placement of males in individual cages prior to mating.
Mated females: Weekly over the periods Days 0-7, 7-14 and 14-20 of gestation, then Days 0-7, 7-14 and 14-21 of lactaton.

- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes
Achieved intake = (Food consumption x dietary ceoncentration)/Body weight
Oestrous cyclicity (parental animals):
Over the 2 weeks prior to pairing for mating the F0 and F1 animals, vaginal lavages were taken early each morning and the stage of oestrus observed was recorded.
Sperm parameters (parental animals):
F0 and F1 parent animals
Testes: weighed individually; left testis fixed in Bouin’s fluid, right testis submitted for sperm evaluation
Epididymides: weighed individually; right cauda epididymis submitted for sperm evaluation
Sperm evaluation:The tip of the cauda epididymis was placed in Medium 199 containing 0.2% BSA and HEPES. The sperm was allowed to ‘swim out’ into the medium. An appropriate dilution of the sperm suspension was examined using a Hamilton Thorne sperm motility analyser; sufficient replicates
to provide 200 motile sperm were assessed. The remaining portion of the cauda epididymis was minced and suspended. Dilutions of that suspension were assessed using a haemocytometer to obtain a total sperm count which was expressed per cauda epididymis and per gram of cauda epididymis.
Litter observations:
The females were allowed to litter normally. The day on which parturition commenced was designated Day 0 of lactation and the duration of
gestation in days was evaluated.
The numbers of live and dead pups born in each litter was recorded as soon as possible after completion of parturition on Day 0 of lactation.
The live pups were sexed, counted and examined for the presence of milk in the stomach and for any externally visible abnormalities daily up to
Day 4 of lactation; they were then sexed, counted and examined for abnormality again on Days 7, 14 and 21 of lactation.
On Day 1 of lactation, the anogenital distance was measured. The live pups were weighed individually on Days 1, 4, 7, 14 and 21 of lactation.
On Day 11 of lactation, the male pups were assessed for nipple or areola retention. Where practicable, any pups that were found dead or were killed during lactation were sexed and appropriately examined as detailed above. Prior to Day 14 of lactation, any externally abnormal decedent pup was preserved, externally normal ones were discarded. On or after Day 14 of lactation, decedent pups were necropsied.
Deficiencies in maternal care were recorded. Points looked for included inadequate construction and cleaning of the nest, pups left scattered and
cold, physical abuse of pups, or apparently inadequate lactation or feeding.

At Day 21 of lactation, a group size of 24 male and 24 female weanlings was constituted, nominally by selection of one male and one female from 24 appropriate litters. For each sex, a pup was randomly selected from that litter. The selected animals were earmarked, although actual
separation from the mother occurred later, at Day 24 of lactation.
Pups that were not selected for post-weaning assessment remained with their mother until termination.
Postmortem examinations (parental animals):
For all F0 and F1 parents
External examination was followed by macroscopic examination of the tissues and organs of the cranial, thoracic and abdominal cavities in situ.
Any gross lesions were described in terms of location, size, shape, colour, consistency, number and any other relevant characteristics.
Representative samples of abnormal tissues were taken and fixed in neutral buffered 10% formalin.

The reproductive tracts of all females were examined for signs of implantation, the number of any implant sites present being recorded.

The following organs were fixed (in neutral buffered formalin unless otherwise specified). Weights were taken, where indicated, prior to fixation:
Ovaries: weighed individually
Uterus (with oviducts and cervix): weighed
Vagina
Testes: weighed individually; left testis fixed in Bouin’s fluid, right testis submitted for sperm evaluation
Epididymides: weighed individually; right cauda epididymis submitted for sperm evaluation
Seminal vesicles and coagulating glands: combined weight recorded
Prostate, Pituitary gland, Brain, Liver, Kidneys (individually), Spleen were all weighed.
Thyroid glands: weighed individually after fixation
Adrenal glands: weighed individually

HISTOLOGY:
The following tissues were processed; sections were cut 4-6 Mm thick, stained with haematoxylin and eosin (H&E) and evaluated by light microscopy:
Ovaries (5 representative sections per ovary), Uterus (with oviducts and cervix), Vagina, Left testis (transverse section), Left epididymis, Seminal
vesicles and coagulating glands, Prostate, Pituitary gland, Adrenal glands.
Additionally, a Periodic Acid Schiff and Haematoxylin (PAS-H) stained section was prepared from the left testis.
Postmortem examinations (offspring):
Offspring (pre-weaning) Found Dead or Killed on or After Day 14 of Lactation: These animals were necropsied. This consisted of external
examination followed by macroscopic examination of the tissues and organs of the cranial, thoracic and abdominal cavities in situ.
Samples of any grossly abnormal tissues were preserved in 10% formalin. The carcasses were then discarded.

Offspring Found Dead or Killed Before Day 14 of Lactation: Where practicable, these animals were sexed, then checked for the presence of milk in
the stomach and for the presence of any externally visible abnormalities. Any abnormal pups were, where practicable, fixed in methylated ethyl
alcohol. Externally normal decedents were discarded.

Weanlings not Selected as Parents of a Subsequent Generation: From each litter, 3 male and 3 female pups (where they were available) were
necropsied. This consisted of external examination followed by macroscopic examination of the tissues and organs of the cranial, thoracic and
abdominal cavities in situ. Sample of any grossly abnormal tissues were preserved in 10% formalin. From one of the 3 pups of each sex, the weights of the brain, spleen and thymus were recorded, and these organs were preserved. The carcasses were then discarded.
Statistics:
All statistical tests were two-sided and performed at the 5% significance level using in house software. Pairwise comparisons were performed against the Control group.
Body weight and food consumption data of males, and of females prior to mating, were subjected to analysis of variance. Where appropriate, a log or square root transformation was used to stabilise the variances. Where transformation failed to stabilise the variances, the Kruskal-Wallis test
was used.
Organ weight data was subjected to analysis of variance, and to one-way analysis of covariance using the terminal body weight as the covariate.
In circumstances where the variances in the ANCOVA remained heterogenous following log or square root transformations or where it was not
possible to perform the F-max test, the untransformed parametric ANCOVA results were reported.
Histopathology data were analysed by Fisher’s Exact Probability test.
For other parameters, interpretation was based on inspection of the individual and group values.
Reproductive indices:
Fertility Index (males) = Number siring a litter/number paired
Fertility Index (females) = Number pregnant/Number paired
Gestation INdex = Number bearing live pups/Number paired
Offspring viability indices:
Birth Index = Total number of pups born (live and dead)/Number of implantation scars
Live Birth index = Number of pups live on Day 0 of lactation/Total number born (live and dead)
Viability index = Number of pups live on Day 4 of lactation/Number live on Day 0
Lactation index = Number of pups live on Day 21 of lactation/Number live on Day 4
Overall Survivial Index = Number of pups live on Day 21 of lactation/Total Number Born (live and dead)

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
during gestation with 2500/7500 ppm diets
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
during gestation with 2500/7500 ppm diets
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Test substance intake: reduced during gestation with weight loss

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS) There were no clinical observations considered to be related to treatment with
p-tertbutylphenol in either generation.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
F0 – adults: At 7500 p.p.m. there was a decrease in weight gain seen in F0 animals over the first week of treatment. The overall weight gain of the
animals over the treatment period (prior to mating) was ca 70% of the Control weight gain, although absolute body weight was generally 80-90% of
the Controls. At 2500 p.p.m. in both sexes and 800 p.p.m. in females, there was a slight but similar decrease in weight gain, compared to the higher dose. There were no clear effects of treatment in males at 800 p.p.m.

F1 - adults: At 7500 p.p.m. the mean body weight of weaned animals (nominal Week 4) was 79% of the Control weight in males and 84% of the Control weight in females. By the end of the treatment period (prior to mating for females) body weight was still ca 80% of the Controls’ body weight; weight gain throughout was lower than that of the Controls (ca 80%). At 2500 p.p.m. and 800 p.p.m. the mean body weight of weaned animals (nominal
Week 4) was 92% of the Controls for males and 91% and 94% for females.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS): In the F0 generation, in both sexes, the achieved intake was lower than expected in the
highest dose during the first week, and it was considered most likely that this reflected the low food consumption over that period. Thereafter the
intake was as expected, with the intake decreasing proportionally as the animals grew.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS):The stages of the oestrus cycles and their mean duration were similar in all groups.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS): There were no effects on the sperm motility, count or morphology at any of the dose levels applied, or in either generation. A lower cauda weight and testes weight in F1 animals was considered most likely to reflect the lower body weight, and did not affect any of the counts.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS): There were no clear effects of treatment on mating performance, fertility or duration of
gestation in either generation.

ORGAN WEIGHTS (PARENTAL ANIMALS): Males: here were no statistically significant effects on organ weights at 7500ppm and 2500 p.p.m., and there were none at 800 p.p.m in either the F0 or F1 males.
Females F0: After covariance analysis, decreases in adrenal gland, ovary and pituitary gland weights were significant (P<0.001).
Females F1: Following covariance analysis a number of organs at 7500 p.p.m. achieved significance, and adrenal gland, brain, kidney, ovaries,
pituitary gland and uterus were lower than Control. There was a significant increase in liver weight. At 2500 p.p.m. adrenal gland and brain weight
were lower than Controls, and achieved significance by analysis of variance and covariance. A slightly higher liver weight achieved significance after covariance analysis. There were no other effects on organ weights at 2500 p.p.m., and there were none at 800 p.p.m for either F0 or F1 females.

GROSS PATHOLOGY (PARENTAL ANIMALS):
There were no findings at necropsy considered to reflect an obvious effect of treatment on either the F0 or F1 adults.
In the F0 generation, there was no clear pattern of effect on follicle type or incidence. However, in the F1 generation, at 7500 p.p.m. there was an
increase in the incidence of primordial follicles with a concurrent decrease in the incidence of growing follicles. At 800 and 2500 p.p.m. the type and incidence of follicles was essentially similar to Controls.

HISTOPATHOLOGY (PARENTAL ANIMALS): In the F0 animals there was an increase in atrophy of the vaginal epithelium with 7/28 animals at
2500 p.p.m. and 12/28 animals at 7500 p.p.m. affected. The severity of the finding was split approximately equally between minimal and mild.
In the F1 animals, this finding was increased in severity and incidence at 7500 p.p.m. The severity was mild in the majority of animals (79%) with a
total of 14/24 animals. However there was no atrophy of the vaginal epithelium recorded at 2500 p.p.m.
There was no atrophy of the vaginal epithelium recorded at 800 p.p.m., and no other findings of note at any dose level in either generation.

OTHER FINDINGS (PARENTAL ANIMALS): In the F0 and F1 generations there were a lower number of implant sites and live pups born at 7500 p.p.m.
The lower numbers of implants may be associated with the lower body weights.

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOEL
Effect level:
70 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
other:
Remarks on result:
other: Generation: P and F1
Key result
Dose descriptor:
LOAEL
Effect level:
200 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: Reduced maternal weights

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
increased mortality in F1 pups at 7500 p.p.m
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
reduced at 2500 and 7500 p.p.m. in F1&F2
Sexual maturation:
effects observed, treatment-related
Description (incidence and severity):
delays of 3 or 4 days after controls
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
detailed below
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed

Details on results (F1)

VIABILITY (OFFSPRING): F0 generation - at 7500 p.p.m. pup survival was poor, particularly over Days 0-4 of lactation when 6 different litters had more than 3 pups dying, and in 2 of these litters all pups died. F1 generation: at 7500 p.p.m. the litter size was clearly smaller than that at any other
level. The survival of these smaller litters was good and exceeded the performance of the Controls. There were no effects of treatment detected at
800 or 2500 p.p.m. There were no effects on sex ratios in either generation.

CLINICAL SIGNS (OFFSPRING): No treatment related effects were seen.

BODY WEIGHT (OFFSPRING): At 7500 p.p.m., in both generations, pup weights at birth were comparable to those of the Controls but the litter size was slightly smaller, and as a result, litter weight was lower than Controls on Day 1 of lactation. After Day 1 of lactation pup weight gain was less than
Control, and by Day 21 of lactation was ca 20% lower than Control weights. Litter weight gain was similarly affected but in the F0 generation this was
also accompanied by a decrease in pup survival. At 2500 p.p.m. pup weight in both generations was lower than Controls from Day 14 of
lactation; with a concurrent decrease in litter weight gain also. There were no effects on litter or pup weight at 800 p.p.m. in either generation.

SEXUAL MATURATION (OFFSPRING): At 7500 p.p.m. vaginal opening and preputial separation occurred 3 and 4 days later than Controls, respectively. The age and body weight at preputial separation or vaginal opening in animals treated with 2500 p.p.m. and 800 p.p.m. p-tertbutylphenol were
essentially similar to Controls.There were no effects of treatment on ano-genital distance, or on nipple/areolar retention.

ORGAN WEIGHTS (OFFSPRING):
Males F0 generation, F1 production
In males treated at 7500 p.p.m. body weight, spleen and thymus weights were all significantly lower than Control.
Brain weight was reduced but did not attain significance. Following covariance analysis none of the findings were evident.
There were no effects on organ weight at 800 or 2500 p.p.m.

Males F1 generation, F2 production
in males treated at 7500 p.p.m., body weight, brain, spleen and thymus weights were all significantly reduced.
Brain weight was reduced but did not attain significance. Following covariance analysis, spleen weight was significantly but slightly lower.
In addition, absolute spleen weight at 2500 p.p.m. was also significantly lower, although this was no longer evident after covariance analysis.
There were no other effects on organ weights at 2500 p.p.m., and there were none at 800 p.p.m.

Females F0 generation, F1 production
In females treated at 7500 p.p.m. all of the same parameters as males were lower, with the brain and thymus slightly reduced at 2500 p.p.m. also.
Following covariance analysis thymus weights at 2500 and 7500 p.p.m. remained marginally lower.
There were no other effects on organ weights at 2500 p.p.m., and there were none at 800 p.p.m.

Females F1 generation, F2 production
In females treated at 7500 p.p.m. body weight, brain and spleen weight were statistically significantly lower than Control; thymus weight was also
lower but did not attain statistical significance. Following covariance analysis, spleen weight was lower than the Control spleen weight, although the
difference did not attain significance. All other weights appeared to be comparable to Controls.
There were no effects on organ weights at 800 or 2500 p.p.m.

GROSS PATHOLOGY (OFFSPRING): At necropsy, there were no findings considered to reflect an obvious effect of treatment on the weanlings of either generation.

HISTOPATHOLOGY (OFFSPRING): There were no findings in the tissue pathology assessed in the offspring that were treatment related.

Effect levels (F1)

Key result
Dose descriptor:
LOAEL
Generation:
F1
Effect level:
600 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Increased pup mortality rate; an increase in the incidence of primordial follicles with a concurrent decrease in the incidence of growing follicles; increase in atrophy of the vaginal epithelium.

Overall reproductive toxicity

Key result
Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
In general, there was no obvious evidence of poorer performance in the second generation compared with the first.
Effects of treatment on reproductive performance were reduced pup weights at 2500 and 7500 p.p.m. in both generations, and increased pup
mortality in F1 pups at 7500 p.p.m.; many of the findings reported appear to be related to maternal size or performance and/ or
direct toxicity. Other findings from this study are those noted in the ovary (follicle changes) and vaginal epithelium atrophy at 2500 and
7500 ppm..
It is therefore considered that the no observed adverse effect level (NOAEL) is 800 ppm pTBP in the diet (ca. 70 mg/kg bw/day intake).
Executive summary:

In a two-generation reproduction study p-tert butyl phenol was administered orally to Sprague Dawley rats (28 males and females F0 generation; 24 males and females F1 generation) at 0, 800, 2500 and 7500 ppm in diet yielding approximate intake levels of 0,70, 200 and 600 mg/kg bw/day.

There were no treatment related clinical signs.

There were no abnormalities noted in the pups associated with treatment.

At 2500 and 7500 p.p.m. there was a decrease in weight gain seen in F0 animals, and in both generations during gestation. In the F1 generation, overall weights of animals at these levels were lower, reflecting the lower weaning weights of these animals.

At 2500 and 7500 p.p.m. food consumption was notably reduced in F0 animals, and in F1 animals at 7500 p.p.m.

The values for achieved intake were generally proportional to the dietary concentrations.

The stages of the oestrus cycles and their mean duration were similar in all groups and there were no clear effects of treatment on mating performance, fertility or duration of gestation in either generation.

In the F0 and F1 generations there were a lower number of implant sites and live pups born at 7500 p.p.m. In the F1 generation, at 7500 p.p.m. there was an increase in the incidence of primordial follicles with a concurrent decrease in the incidence of growing follicles.

At 7500 p.p.m., there was an increase in atrophy of the vaginal epithelium in F0 and F1.

There were no effects on the sperm motility, count or morphology at any of the doseblevels applied, or in either generation.

There was no necropsy findings considered to reflect an obvious effect of treatment on either the F0 or F1 adults, or on the pups of either generation.

In general, there was no obvious evidence of poorer performance in the second generation compared with the first.

No effects of treatment were observed at 800ppm.

The NOAEL is 800 ppm (70 mg/kg bw/day in females).

This study is acceptable and satisfies the guideline requirement for a two-generation reproductive study (OPPTS 870.3800); OECD 416 in rat.