Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
17 May - 08 Jul 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA OPPTS 870.3550, Reproductive/Developmental Toxicity Screening Test. July, 2000
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): tris(hydroxymethyl)aminomethane, 2-amino-2-(hydroxymethyl)-1,3-propanediol, tromethamine
- Analytical purity: 99.9% (with 0.018% wt. water in batch XK0731LA1C and 0.021% wt. water in batch XK1231LA1C)
- Purity test date: 2011
- Lot/batch No.: XK0731LA1C and XK1231LA1C
- Storage condition of test material: at room temperature

Test animals

Species:
rat
Strain:
other: Crl:CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS- Source: Charles River Laboratories Inc., Portage, Michigan, USA- Age at study initiation: (P) approximately 8 weeks- Weight at study initiation: (P) Males: 255.2 ± 4.9-256.6 ± 6.2 g; Females: 190.6 ± 8.6- 194.6 ± 8.5 g (mean ± SD) - Fasting period before study: no- Housing: animals were housed individually in stainless steel cages, except during breeding and during the littering phases; during breeding, one male and one female were housed per cage; during littering, dams and their litters were housed in plastic cages provided with ground corncob nesting material from approximately GD 19 until termination. Cages had wire mesh floors and were suspended above catch pans. Non-woven gauze was placed in the cages to provide a cushion from the flooring for rodent feet and also provided environmental enrichment. In order to better visualize copulation and plugs, gauze was not placed in cages during the breeding phase. - Diet: LabDiet Certified Rodent Diet #5002 (PMI Nutrition International, St. Louis, Missouri) in meal form, ad libitum- Water: tap water, ad libitum- Acclimation period: at least 7 daysENVIRONMENTAL CONDITIONS- Temperature (°C): 22 ± 1 (maximum permissible excursion of ± 3 °C)- Humidity (%): 40-70- Air changes (per hr): 12-15, on average- Photoperiod (hrs dark / hrs light): 12/12IN-LIFE DATES: From: 17 May 2011 To: 15 Jun 2011 (males); 30 Jun and 08 Jul 2011 (females)

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The test material was mixed with deionised water to reach a dose volume of 4 mL/kg bw; calculated according to the latest body weight, and the solution was adjusted to pH 9. Dose solutions were prepared periodically during the study period based on stability data.VEHICLE- Concentration in vehicle: 25, 75 and 250 mg/mL- Amount of vehicle (if gavage): 4 mL/kg bw
Details on mating procedure:
- M/F ratio per cage: 1/1- Length of cohabitation: until pregnancy was confirmed or up to two weeks- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy- After successful mating each pregnant female was caged (how): individually
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses by HPLC/ELSD were performed to determine the concentration of the test material of all dosing solutions from the first mix of the main study prior to the start of dosing. The low- and high-dose solutions from the first mix of the main study were analyzed to confirm homogeneous distribution of the test material concurrent with dose confirmation. The stability was measured on Day 0, 1, 6, 15 and 22. The stability results were 93.4-112.9% of target concentration with RDS up to 18.1% on Day 0; and 81.7-109.4% of target concentration with RDS up to 13.9% on Day 22. The concentration and homogeneity of the dose solutions was 91.4-109.1% of target concentration, with RSD of up to 12.5% (N = 6). Quantitation was performed using external standard calibration; the accuracy of the standards was 94.6-110% (mean) of the nominal values. The large RSDs were explained by the use of an external standard calibration and the ELSD, which both tend to give a larger RSD.
Duration of treatment / exposure:
(P) Males: 29 days (14 days prior to mating, 14 days during mating, up to and including Day 29) (P) Females: up to 54 days (14 days prior to mating, up to 14 days during mating, approximately 22 days during gestation, 4 days during lactation)
Frequency of treatment:
Daily, 7 days/week
Details on study schedule:
- Age at mating of the mated animals in the study: approximately 10 weeks
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: a range-finding study was performed in which 3 rats/sex/dose were administered 0, 250, 500, 750 or 1000 mg/kg bw/day via gavage for 14 days. All the animals survived to scheduled termination and there were no treatment-related clinical signs. The body weights and feed consumption were comparable between the control and treatment groups, and no effects were noted on organ weights or during gross necropsy. As no effects were observed at the highest dose level of 1000 mg/kg bw/day and this is the limit dose defined in the EPA OPPTS 870.3550 guideline, dose levels of 100, 300 and 1000 mg/kg bw/d were selected for the main study. - Other: the pH of the test solution was adjusted to 9 prior to dosing

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes- Time schedule: at least twice daily during the study period for all animals, including dams and their litters- Cage side observations included, but were not limited to: morbidity, mortality, decreased/increased activity, repetitive behaviour, vocalization, incoordination/limping, injury, neuromuscular function (convulsion, fasciculation, tremor, twitches), altered respiration, blue/pale skin and mucous membranes, severe eye injury (rupture), alterations in fecal consistency, and fecal/urinary quantity DETAILED CLINICAL OBSERVATIONS: Yes- Time schedule: daily. Animals were observed for abnormalities in the eyes, urine, feces, gastrointestinal tract, extremities, movement, posture, reproductive system, respiration, skin/hair-coat, and mucous membranes, as well as an assessment of general behaviour, injuries, or palpable mass/swellings. Females were observed for signs of parturition from around gestation day 20.BODY WEIGHT: Yes- Time schedule for examinations: for all animals, at least once during the pre-exposure period and on the first day of dosing (Day 0). Males were also weighed weekly during the study period. Females were weighed weekly during the pre-mating and mating period; on gestation day (GD) 0, 7, 14, 17 and 20; females that delivered litters were weighed on lactation day 1 and 4; females that failed to mate or deliver a litter were weighed at least weekly until termination.FOOD CONSUMPTION:- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes. Feed consumed was determined weekly during the two week pre-breeding period for males and females by weighing feed crocks at the start and end of a measurement cycle. Feed consumption was not measured for males or females due to co-housing during breeding. Following breeding, feed consumption was not measured for males. For females during gestation, feed consumption was measured on GD 0, 7, 14, and 20. After parturition, feed consumption was measured on lactation day 1 and 4. Feed consumption was not recorded for females that failed to mate or deliver a litter. - Compound intake calculated as time-weighted averages from the consumption and body weight gain data: NoWATER CONSUMPTION: No
Sperm parameters (parental animals):
Parameters examined in P male parental generations:- all males: testis weight, epididymis weight
Litter observations:
STANDARDISATION OF LITTERS- Performed on day 4 postpartum: no, pups were terminated on lactation day 4PARAMETERS EXAMINEDThe following parameters were examined in F1 offspring:number and sex of pups, stillbirths, live births, presence of gross anomalies, body weight (lactation day 1 and 4), physical or behavioural abnormalities, number of live and dead pups (lactation day 0, 1 and 4), clinical observations (daily) GROSS EXAMINATION OF DEAD PUPS:yes, for external abnormalities; possible cause of death was not determined for pups born or found dead.
Postmortem examinations (parental animals):
SACRIFICE- Male animals: all surviving animals were sacrificed after 29 days of exposure, in a fasted state- Maternal animals: all surviving animals were sacrificed between lactation day 5 and 8, or at least 24 days after the end of the mating period for females not producing a litter, in a fasted stateGROSS NECROPSY- Gross necropsy consisted of external examination of tissues and orifices, in situ examination of the eyes, and internal examinations including the cervical, thoracic, and abdominal viscera.Samples of the following tissues/organs were collected from all the animals and preserved in neutral, phosphate-buffered 10% formalin: kidneys, liver, pituitary, gross lesions, cervix, coagulating glands, mammary gland (females), ovaries, oviducts, prostate, seminal vehicles, uterus, vagina. The testes and epididymides were fixed in Bouin’s. Transponders were removed and placed in jars with the tissues. The uteri of all females in control and treatment groups were stained with an aqueous solution of 10% sodium sulfide stain (Kopf et al., 1964) for approximately one minute and were examined for the presence and number of implantation sites, prior to preservation. HISTOPATHOLOGY / ORGAN WEIGHTSThe weights of the epididymides, kidneys, liver, and testes were recorded for all the animals in the control and treatment groups. Histological examinations were performed on the preserved tissues/organs from the animals in the control and high-dose groups. A qualitative assessment of the stages of spermatogenesis was made of the testes in control and high-dose males by examining the relationship between spermatogonia, spermatocytes, spermatids, and spermatozoa (cycle of spermatogenesis) as seen in cross sections of the seminiferous tubules by microscopic evaluation; and by examination of sections of both testes for the presence of degenerative changes (e.g., vacuolation of the germinal epithelium, a preponderance of Sertoli cells, sperm stasis, inflammatory changes, mineralization, and fibrosis)
Postmortem examinations (offspring):
SACRIFICE- The F1 offspring was sacrificed at 4 days of ageGROSS NECROPSY- Gross necropsy consisted of external examinations of the pupsHISTOPATHOLOGY / ORGAN WEIGTHSNo histopathological examinations were performed; no organs weights were recorded
Statistics:
Parental body weights and gestation and lactation body weight gains, litter mean body weights, feed consumption, and organ weights (absolute and relative) was first evaluated by Bartlett's test (alpha = 0.01; Winer, 1971) for equality of variances. Based upon the outcome of Bartlett's test, either a parametric (Steel and Torrie, 1960) or non-parametric (Hollander and Wolfe, 1973) analysis of variance (ANOVA) was performed. If the ANOVA was significant at alpha = 0.05, a Dunnett's test (alpha = 0.05; Winer, 1971) or the Wilcoxon Rank-Sum (alpha = 0.05; Hollander and Wolfe, 1973) test with Bonferroni's correction (Miller, 1966) was performed. Feed consumption values were excluded from analysis if the feed is spilled or scratched. Gestation length, average time to mating, and litter size were analyzed using a nonparametric ANOVA. If the ANOVA was significant, the Wilcoxon Rank-Sum test with Bonferroni's correction was performed. Statistical outliers (alpha = 0.02) were identified by the sequential method of Grubbs (1969) and only excluded from analysis for documented, scientifically sound reasons. The mating, conception, fertility and gestation indices were analyzed by the Fisher exact probability test (alpha = 0.05; Siegel, 1956) with Bonferroni's correction. Evaluation of the neonatal sex ratio on postnatal day 1 was performed by the binomial distribution test (alpha = 0.05; Steel and Torrie, 1960). Gender of pups found dead on postnatal day 0 was included in sex ratio calculations. Survival indices, post-implantation loss, and other incidence data among neonates was analyzed using the litter as the experimental unit by the censored Wilcoxon test (alpha = 0.05; Hollander and Wolfe, 1973) as modified by Haseman and Hoel (1974) with Bonferroni’s correction. Non-pregnant females, females with resorptions only, or females found to be pregnant after staining of their uteri were excluded from the appropriate analyses.
Reproductive indices:
Calculated for all doses levels:Female mating index = (No. females with evidence of mating/No. paired) x 100Male mating index = (No. males with evidence of mating/No. paired) x 100Female conception index = (No. females with evidence of pregnancy/No. mated) x 100Male conception index = (No. males siring a litter/No. mated) x 100Female fertility index = (No. females with evidence of pregnancy/No. paired) x 100Male fertility index = (No. males siring a litter/No. paired) x 100Gestation index = (No. females delivering a viable litter/No. females delivering a litter) x 100Post-implantation loss = (No. implants – No. viable offspring)/(No. implants) x 100
Offspring viability indices:
Calculated for all dose levels:Gestation survival index = percentage of delivered pups alive at birthDay 1 or 4 pup survival index = (No. viable pups on day 1 or 4/No. born live) x 100

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
1000 mg/kg bw/day: thickening of the limiting ridge, local irritation effect
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
300 mg/kg bw/day: hyperplasia of the epithelium of the limiting ridge (males), inflammation of the submucosa, glandular stomach, local irritation effect
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)There was no mortality during the study period. No treatment-related clinical signs were observed during the study period.BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)No significant differences in body weight or body weight gain were observed between the male control and treatment groups. In females, no significant differences in body weight gain were noted between the control and treatment groups. A statistically significant increase in body weight gain was observed in the female mid-dose group during lactation day 1-4, compared to the control group. This effect is considered to be incidental, as no effect was noted in the female high-dose group. There were no significant differences in feed consumption between the control and treatment groups during the study period. TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)The test substance was administered by gavage daily, with doses based on the body weight, ensuring an accurate dosing of the animals.REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)There were no effects on the testis weight or epididymis weight in the control or treatment groups.REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)No treatment-related effects were noted on the male/female mating index, male/female conception index, male/female fertility index, gestation index, time to mating, gestation period, and post-implantation loss (see Table 1). ORGAN WEIGHTS (PARENTAL ANIMALS)There was no significant difference in organ weights between the control and treatment groups.GROSS PATHOLOGY (PARENTAL ANIMALS)10/10 females and 10/10 males in the high-dose groups had a thickened limiting ridge of the stomach (see Table 2). The limiting ridge is a thin protrusion from the forestomach that demarcates the forestomach from the glandular stomach in rats. The effects are considered to be caused by a local irritation effect of the test substance and are therefore treatment-related. However, as humans do not have a forestomach and limiting ridge, the findings are of limited relevance to humans. The gross pathology examinations did not reveal any treatment-related findings in the reproductive organs.HISTOPATHOLOGY (PARENTAL ANIMALS)Very slight to slight hyperplasia of the stratified squamous epithelium was observed at the limiting ridge of the stomach of 1, 0, 4 and 10 male, and in 2, 0, 2 and 10 female rats in the control, low-, mid- and high-dose group, respectively (see Table 2). In addition, subacute to chronic inflammation of the submucosa of the glandular stomach was noted in at least 1 animal in all groups; classified as very slight or slight. A clear treatment-related effect was seen only in the cases classified as slight; where 0, 0, 3 and 9 males, and 0, 0, 1 and 4 females in the control, low-, mid- and high-dose group, respectively, were affected. This is considered to be a local irritating effect of the test substance, which was administered in a relatively large volume. As humans do not have a limiting ridge and forestomach, the effect is considerd to have limited toxicological relevance to humans. There were no treatment-related histopathological findings in the reproductive organs.

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
reproduction
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Reproductive effects
Remarks on result:
other:
Remarks:
No reproductive effects for offspring growth and survival at any dose level
Key result
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects observed up to and including the highest dose level
Remarks on result:
other:
Remarks:
No effect observed up to and including the highest dose level
Key result
Dose descriptor:
NOAEL
Remarks:
local
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: histopathology; hyperplasia of the limiting ridge, inflammation of the submucosa of the glandular stomach. This effect is not relevant to humans.
Remarks on result:
other:
Remarks:
Not relevant for humans

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined

Details on results (F1)

VIABILITY (OFFSPRING)There were no significant differences in gestation survival index or viability between the control and treatment groups (see Table 1). Due to the death of several pups in high-dose litters, the survival index for this dose group was slightly lower than the survival index for the remaining groups, at 99.3% on lactation day 1 and 97.8% on lactation day 4, compared to 100% for all other groups on lactation day 1 and 4. However, it remained within the historical data range (97.7-100.0%, see Table 3).CLINICAL SIGNS (OFFSPRING)No treatment-related clinical signs were observed in the control or treatment groups.BODY WEIGHT (OFFSPRING)There were no significant differences in body weight between the control and treatment groups on lactation day 1 and 4. GROSS PATHOLOGY (OFFSPRING)No treatment-related effects were noted during the gross pathological examination in any group.OTHER FINDINGS (OFFSPRING)The sex ratio was statistically significantly changed in the control group, compared to all the treatment groups. As only the control group was affected, this is considered to be an incidental observation (see Table 1).

Effect levels (F1)

Key result
Dose descriptor:
other:
Remarks:
No effects observed
Generation:
F1
Effect level:
> 100 - <= 1 000 mg/kg bw/day
Based on:
test mat. (total fraction)
Sex:
male/female
Basis for effect level:
viability
mortality
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no
Lowest effective dose / conc.:
1 000 mg/kg bw/day (nominal)
System:
other: body weight and gross external abnormalities
Organ:
not specified

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Any other information on results incl. tables

Table 1: Reproduction performance and developmental parameters

Observations 

Dose (mg/kg bw/day)

 

Control

100

300

1000

Pairs started (N)

10

10

10

10

Male mating index, % (N)

100.0 (10/10)

100.0 (10/10)

100.0 (10/10)

100.0 (10/10)

Female mating index, % (N)

100.0 (10/10)

100.0 (10/10)

100.0 (10/10)

100.0 (10/10)

Time to mating, days (mean ± SD)

2.0 ± 0.9

3.7 ± 3.2

2.7 ± 1.8

2.9 ± 1.1

Male fertility index, % (N)

80.0 (8/10)

100.0 (10/10)

90.0 (9/10)

100.0 (10/10)

Female fertility index, % (N)

80.0 (8/10)

100.0 (10/10)

90.0 (9/10)

100.0 (10/10)

Post-implantation loss, % (mean ± SD)

8.88 ± 14.93

9.79 ± 13.64

4.18 ± 4.44

4.89 ± 5.25

Gestation length, days (mean ± SD)

21.6 ± 0.5

21.5 ± 0.5

21.4 ± 0.5

22.0 ± 0.5

Gestation index, % (N)

100.0 (8/8)

100.0 (10/10)

100.0 (9/9)

100.0 (10/10)

Gestation survival index, % (N)

100.0 (110/110)

100.0 (138/138)

99.3 (138/139)

100.0 (138/138)

Day 1 survival index, % (N)

100.0 (110/110)

100.0 (138/138)

100.0 (138/138)

99.3 (137/138)

Day 4 survival index, % (N)

100.0 (110/110)

100.0 (138/138)

100.0 (138/138)

97.8 (135/138)

Live pups/dam at birth (mean ± SD)

13.8 ± 2.5

13.8 ± 2.8

15.3 ± 1.4

13.8 ± 3.5

Live pups/dam lactation day 1 (mean ± SD)

13.8 ± 2.5

13.8 ± 2.8

15.3 ± 1.4

13.7 ± 3.6

Live pups/dam lactation day 4 (mean ± SD)

13.8 ± 2.5

13.8 ± 2.8

15.3 ± 1.4

13.5 ± 3.5

Pups born dead

0/110

0/110

1/139

0/138

Sex ratio Day 1, male:females

64:36*

49:51

47:53

50:50

Pup body weight lactation day 1, g (mean ± SD)

- male

- female

 

 

7.6 ± 0.7

7.1 ± 0.7

 

 

7.5 ± 0.8

7.1 ± 0.7

 

 

7.1 ± 0.6

6.6 ± 0.6

 

 

7.8 ± 1.0

7.4 ± 1.0

Pup body weight lactation day 4, g (mean ± SD)

- male

- female

 

 

10.8 ± 1.3

10.2 ± 1.2

 

 

10.7 ± 1.3

10.2 ± 1.3

 

 

10.2 ± 0.8

9.6 ± 0.8

 

 

11.2 ± 2.0

10.7 ± 2.1

* p = 0.05

 

 

Table 2: Results of clinical parameters

Sex

Males

Females

Dose (mg/kg bw/day)

0

100

300

1000

0

100

300

1000

Stomach, No. examined

10

10

10

10

10

10

10

10

Hyperplasia, with inflammation, epithelium, limiting ridge

 

 

 

 

 

 

 

 

- Very slight

 

1

0

4

3

2

0

2

5

- Slight

0

0

0

7

0

0

0

5

Subacute to chronic inflammation, glandular submucosa, multifocal

 

 

 

 

 

 

 

 

- Very slight

3

2

3

1

2

1

6

5

- Slight

0

0

3

9

0

0

1

4

 

Table 3: Historical control data, pup survival index (%) post-natal day 4

Study No.

1

2

3

4

5

6

7

8

9

Year reported

2007

2007

2008

2009

2010

2010

2010

2011

2011

Post-natal day 4

99.4

98.8

97.7

99.4

95.4

99.4

99.4

97.7

100.0

Applicant's summary and conclusion

Conclusions:
2-amino-2-(hydroxymethyl)propane-1,3-diol had no effect on reproductive performance.
Executive summary:

The potential effects of tris(hydroxymethyl)aminomethane on reproductive function, and prenatal/early neonatal growth and survival of the offspring, were investigated in rats following repeated gavage administration, according to OECD Guideline 421. Groups of 10 male and 10 female Crl:CD(SD) rats were administered the test material daily, by gavage, at dose levels of 0 (control), 100, 300, or 1000 mg/kg/day. Female rats were dosed once daily for approximately two weeks prior to breeding, through breeding (up to two weeks), gestation (three weeks), and lactation (four days) up to termination. Male rats were dosed for two weeks prior to breeding and continuing through breeding (two weeks) until necropsy (test day 30). Effects on gonadal function, mating behavior, conception, development of the conceptus, parturition and early postnatal growth and survival were evaluated. In addition, a gross necropsy and histopathology of the adults were conducted with an emphasis on organs of the reproductive system.

Treatment-related parental toxicity was limited to point of contact irritation in the stomach of animals given 300 and 1000 mg/kg/day. Treatment-related gross pathological findings were limited to a thickened limiting ridge of the forestomach in males and females in the 1000 mg/kg/day group. Treatment-related histopathological changes consistent with point of contact irritation in the stomach were observed in the 300 and 1000 mg/kg/day groups. These findings consisted of: 1) hyperplasia of the stratified squamous epithelium at the limiting ridge of the forestomach with inflammation in males given 300 mg/kg/day (very slight) or males and females given 1000 mg/kg/day (very slight to slight) and 2) subacute to chronic, multifocal inflammation of the submucosa of the glandular stomach of males (slight) or females (very slight to slight) given 300 or 1000 mg/kg/day. All these treatment-related changes were interpreted to be localized irritation effects on the stomach due to repeated oral gavage of the test material. There were no treatment-related histopathologic changes in the stomach of males and females given 100 mg/kg/day. There were no effects on any parameter of reproductive performance or offspring survival at any dose level tested. Based on the histopathologic stomach effects, the no-observed-effect level (NOEL) for parental toxicity was 100 mg/kg/day. The NOEL for reproductive effects or offspring growth and survival was 1000 mg/kg/day, the highest dose tested.