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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:
25.02.1988 - 29.11.1988
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Referenceopen allclose all

Reference Type:
publication
Title:
A two-generation reproduction study with hydroquinone in rats.
Author:
Blacker AM, Schroeder RE, English JC, Murphy SJ, Krasavage WJ & Simon GS
Year:
1993
Bibliographic source:
Fundam Appl Toxicol 21, 420 - 424
Reference Type:
study report
Title:
Unnamed
Year:
1989
Report date:
1989

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
EPA OTS 798.4700 (Reproduction and Fertility Effects)
Deviations:
no
GLP compliance:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
Hydroquinone
EC Number:
204-617-8
EC Name:
Hydroquinone
Cas Number:
123-31-9
Molecular formula:
C6H6O2
IUPAC Name:
hydroquinone
Details on test material:
Purity: > 99 % (photographic grade)
Physical state: white crystalline powder
LOT: 71541
Supplier: Tennessee Eastman Co., Kingsport

Test animals

Species:
rat
Strain:
other: CD Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
Age at receipt: 28 d
Acclimation period (P0): 14 d
Age at initiation of treatment (P0): 42 d
Age at initiation of mating (P0): 112 d
Temperature (°C): 14 - 27
Humidity (%): 40 - 88
Photoperiod (hrs dark / hrs light): 12 / 12
Sprague-Dawley rats (Charles River CD strain, Charles River Laboratories, Inc., Portage, MI) were assigned randomly to groups (30 rats/sex/group) 1 week prior to initiation of treatment. These animals were designated as FO parents.
All animals were individually housed during the study except during mating (1 male and 1 female/cage) and lactation (dam and litter/cage).
Tap water and Purina certified rodent chow, No. 5002, were provided ad lib.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
Applied volume dose: 5 mL/kg
Details on mating procedure:
P0 and P1 parents were dosed for at least 70 days prior to mating, throughout the mating and lactation periods, and until scheduled euthanasia. Body weights and feed consumption of parental animals were measured at approximately weekly intervals throughout the study. At mating, one male and one female within the same treatment group were housed together nightly until evidence of mating (presence of copulatory plug or sperm in a vaginal smear) was observed. If after 7 days no evidence of mating was noted, the female was reassigned to another male within the group for a second 7 day period. The day on which evidence of mating was observed was considered to be day 0 of gestation. Females were allowed to litter, and day 0 of lactation was the day on which all pups in a litter were delivered.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dosing solutions were analyzed by HPLC.
Dosing solutions were prepared daily by weighing an appropriate amount of HQ into amber glass jars and then adding degassed distilled water immediately prior to dosing. Analytical verification, performed periodically during the study, demonstrated that the dosing solutions were stable throughout the dosing period and averaged 99.4, 101, and 100 % of target concentrations for the low, mid, and high doses, respectively.
Duration of treatment / exposure:
P0: 10-week premating period and daily treatment during the ensuing mating period. Males continued to be treated daily until sacrifice, while mated females continued to be treated daily during ensuing gestation and laction periods until sacrificed.
P1: 11-week premating period and throughout the ensuing mating, gestation and lactation period until sacrificed.
Study was terminated at day 278
Frequency of treatment:
Dosing once daily on 7 d per w
Details on study schedule:
F1 and F2 litters were evaluated for number of pups (live and dead) and sex of each pup, pup weights, and gross external abnormalities on Days 0, 4, 7, 14, and 21 of lactation. On Day 4 of lactation, litters were culled to 8 pups/litter, if necessary, with sex distribution equalized as close as possible (4 pups/sex/litter). At weaning of the F1 litters on Day 24 of lactation, at least 1 pup/sex/litter was chosen, if possible, for the F1 parental generation which would be used to produce the F2 generation (30 animals/sex/group). Treatment of the animals selected for F1 parents was initiated at 25 days of age. During mating of the P1 animals, care was taken to avoid brother-sister matings.
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
15 mg/kg bw/day (nominal)
Dose / conc.:
50 mg/kg bw/day (nominal)
Dose / conc.:
150 mg/kg bw/day (nominal)
No. of animals per sex per dose:
30 m / 30 f
Control animals:
yes, concurrent vehicle
Details on study design:
Dose selection rationale:
Doses of 15, 50, and 150 mg/kg/d were selected based upon previous studies which suggested the high dose would produce modest parental toxicity and the low dose would be a NOEL for all effects. The high dose was selected based upon overt toxicity (tremors, convulsions, mortality) observed at 300 mg/kg/d (oral dosing in 5 days per week) in male rats in a dominant lethal study.

In this study the following parameters were examined:
Gonadal function, mating behavior, conception, parturition, lactation, weaning, growth and development of offspring

Examinations

Postmortem examinations (parental animals):
P0 and P1 parents were exsanguinated under ethyl ether anesthesia. Males were euthanatized approximately 3 to 4 weeks after completion of the mating period, and females were euthanatized after weaning of the last litter. All parental animals were necropsied and the following tissues were preserved in 10 % neutral buffered formalin: for males, seminal vesicles, prostate gland, pituitary gland, and gross lesions; testes and epididymides were fixed initially in Bouin's solution for 24 – 48 hrs; for females, vagina, uterus, ovaries, pituitary gland, and gross lesions. After fixation, tissues for all P0 and P1 control and high-dose animals were embedded in paraffin, cut at 6 µm, mounted on glass slides, stained with hematoxylin and eosin, and examined by light microscopy. All gross lesions from all parental animals were processed and examined as well. Reproductive tissues for any P0 or P1 parents in the low- and mid-dose groups that failed to produce a litter were also processed and examined by light microscopy.
Postmortem examinations (offspring):
All pups culled on day 4 of lactation, F1 pups not selected as parental animals, and F2 pups were euthanatized and given a gross external and internal examination. Intact pups found dead at birth or during lactation were also given a gross external and internal examination.
Statistics:
Quantitative continuous variables were analyzed first using Bartlett's test for equal variances with a p <= 0.01 for statistical significance. Data with homogeneous variances (i.e., parametric data) were then analyzed by a one-way ANOVA using the F distribution for significance followed, when necessary (p <= 0.05), by Dunnett's test for pairwise comparisons. For data with heterogeneous variances, the Kruskal-Wallis test was used and if differences were indicated (p <= 0.05), a summed rank test (Dunn) was employed for pairwise comparisons. Trends in dose levels were also analyzed using standard regression techniques with a test for trend and lack of fit for parametric data and Jonckheere's test for nonparametric data. For pairwise comparisons and trend analyses, the probability value of p <= 0.05 was used as the criterion for statistical significance. Statistical analysis of frequency data was performed using x2 to test for goodness of fit followed by Fisher's exact test for pairwise comparisons with the significance level corrected by the Bonferroni method. Trend analysis was performed by Armitage's test for linear trend. A probability value of p <= 0.05 was used as the criterion for statistical significance.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Mild tremors were observed in several P0 adults (m/f) at 150 mg/kg/d and in a single P0 male at 50 mg/kg/d. Tremors were exhibited infrequently and shortly after dosing. No other clinical signs of toxicity were noted in parental animals.
Mortality:
mortality observed, non-treatment-related
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
no effects observed

Reproductive function / performance (P0)

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

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Remarks:
parental toxicity
Effect level:
15 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
Dose descriptor:
NOAEL
Remarks:
reproductive effects
Effect level:
150 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive performance

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Mild tremors were observed in several P1 adults (m/f) at 150 mg/kg/d.
Mortality:
mortality observed, non-treatment-related
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Trend analysis revealed a statistically significant (p <= 0.05) dose-related decrease in weight gain suggesting a possible treatment-related effect for the P1 parental males at doses >= 50 mg/kg.
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
no effects observed

Reproductive function / performance (P1)

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

Effect levels (P1)

open allclose all
Dose descriptor:
NOAEL
Effect level:
15 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive performance

Results: F1 generation

General toxicity (F1)

Mortality / viability:
mortality observed, non-treatment-related
Body weight and weight changes:
no effects observed

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
150 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
body weight and weight gain
other: sex ratio

Results: F2 generation

General toxicity (F2)

Mortality / viability:
mortality observed, non-treatment-related
Body weight and weight changes:
no effects observed

Effect levels (F2)

Dose descriptor:
NOAEL
Generation:
F2
Effect level:
150 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
body weight and weight gain
other: sex ratio

Overall reproductive toxicity

Reproductive effects observed:
no

Any other information on results incl. tables

Evidence of slight parental toxicity (occasional occurrence of transient tremors) was observed for some P0 and P1 animals. Mild tremors were observed in several P0 and P1 adults (m/f) at 150 mg/kg/d and in a single P0 male at 50 mg/kg/d. Tremors were exhibited infrequently and shortly after dosing. No tremors were reported for control or low dose animals. No other clinical signs of toxicity were noted in parental animals. Food consumption and survival at all dose levels were comparable to control values throughout the study. No statistically significant differences in body weights were observed for P0 or P1 parental females throughout the study. Body weights for P0 males were also comparable to those of controls throughout the study. Statistically significant differences in body weights were noted for the P1 parental males at several intervals during the pre-mating, mating, and post-mating periods. P0 male mean body weight gains during pre-mating were 335, 325, 341 and 315 g at 0, 15, 50, and 150 mg/kg/d, respectively. P1 male mean body weight gains at 0, 15, 50, and 150 mg/kg/d were 377, 374, 358 and 354 g, respectively, during pre-mating and 43, 35, 42 and 39 g, respectively, during mating and post-mating periods. Trend analysis revealed a statistically significant (p <= 0.05) dose-related decrease in weight gain suggesting a possible treatment-related effect for the P1 but not the P0 parental males. Reproductive performance was not adversely affected by administration over two generations. Pregnancy rates and male fertility indices were comparable between treated and control groups for both P0 and P1 parents. For the P0 adults, two control, five low-dose, seven mid-dose, and one high-dose female(s) failed to mate with the first male and were reassigned to another male. Of these females, only two low-dose and two mid-dose failed to mate with the second male. For P1 adults, three control, eight low-dose, nine mid-dose, and eight high-dose females failed to mate with the first male and were reassigned to another male. Of these females, only one control, one low-dose, six mid-dose, and two high-dose failed to mate with the second male. In general, gestation length for control and treated females was 20 – 23 days. All females showing evidence of parturition delivered at least 1 live pup. Mean numbers of pups per litter, sex ratio, and pup viability at birth and survival through lactation were similar among all groups for F1 and F2 litters. The total number of dead pups found at birth or during the lactation period were 13, 13, 9 and 10 for F1 litters and 17, 13, 26 and 23 for F2 litters at respective doses of 0, 15, 50, and 150 mg/kg/d. Statistically significant increases in pup weights were noted for F2 pups at 15 and 50 mg/kg on postnatal day 0 (weights for these pups were comparable to those of control at subsequent time points).

Gross postmortem examination gave no treatment-related lesions in either P0 and P1 parental animals or F1 and F2 pups. Further, no microscopic lesions attributable to the treatment were found in tissues from F0 or F1 parental animals subjected to histopathologic examination.

Applicant's summary and conclusion

Conclusions:
The results of this two-generation study with rats gave no indication for reproductive toxicity.
Executive summary:

In this study performed according to EPA OTS 798.4700, the effects of hydroquinone on reproductive performance and fertility in a two-generation study with CD Sprague-Dawley rats (one litter per generation) were studied. HQ was administered in an aqueous solution by gavage at doses of 0, 15, 50 and 150 mg/kg bw/d (30 m / 30 f per group). P0 and P1 parental animals were dosed daily for at least 10 weeks prior to cohabitation, during cohabitation, and until scheduled termination. At all dose levels tested, no adverse effects were observed on feed consumption, survival, or reproductive parameters for the P0 or P1 parental animals. Mild, transient tremors were observed shortly after dosing at 150 mg/kg/d in several P0 and P1 parental animals and in a single P0 male at 50 mg/kg/d. These tremors occurred infrequently and were considered to be due to an acute stimulatory effect of HQ on the nervous system. Body weights for F0 and F1 parental females were similar between all dose groups throughout the study. Body weights for P0 parental males were also comparable to those of control throughout the study. Statistically significant differences in body weights were noted for the P1 parental males in the 50 and 150 mg/kg/d dose groups at several intervals during the pre-mating, mating, and post-mating periods. No treatment-related effects on pup weight, sex distribution, or survival were noted for pups of either generation. Upon postmortem examination, no treatment-related gross lesions were observed in either the P0 or P1 parental animals or their weanlings. Histopathological examination of reproductive tissues and pituitary glands from high-dose P0 and P1 parental animals did not reveal any changes related to treatment. Thus, no adverse effects on reproduction or fertility were observed in either generation at any dose level, and the results are indicating that HQ is not a selective reproductive toxicant. The NOEL values for general and reproductive toxicity are 15 and 150 mg/kg/d, respectively.