Registration Dossier

Administrative data

Endpoint:
two-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
GLP study according to US national standard, which was similar to the OECD TG 416, however with the following deviations. Organ weights and histopathology parameters were not assessed. However, the main purpose of a two-generation reproduction study is to identify effects on reproductive parameters and this purpose is fully met by the present study. Organ weights are determined in other repeated-dose studies and histopathology of reproductive organs is also common practice in these studies. No remarkable findings were made in the various repeated-dose studies. Sperm parameters were also not assessed, but the fact that reproductive performance was not impaired in the present study allows the conclusion that the number and motility of spermatozoa was not reduced by the test compound. Thus, despite o the deviation reported above, the study can be considered as valid for assessment of potential hazards to reproduction by triclosan.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1988
Report Date:
1988

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
EPA OPP 83-4 (Reproduction and Fertility Effects)
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Deviations:
yes
Remarks:
see below
Principles of method if other than guideline:
Deviations of the method:
- The mating period was up to 31 days (recommended: two weeks).
- Organ weights and histopathology parameters were not assessed.
- However, the main purpose of a two-generation reproduction study is to identify effects on reproductive parameters and this purpose is fully met by the present study. Organ weights are determined in other repeated-dose studies and histopathology of reproductive organs is also common practice in these studies. No remarkable findings were made in the various repeated-dose studies. - Sperm parameters were also not assessed, but the fact that reproductive performance was not impaired in the present study allows the conclusion that the number and motility of spermatozoa was not reduced by the test compound. - The present study is therefore valid for assessment of potential hazards to reproduction by triclosan.
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): FAT 80'023
- Physical state: white powder with lumps
- Analytical purity: ≥ 99%
- Lot/batch No.: 5202110
- Stability under test conditions: stability and homogeneity in diet was analytically verified

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: the Sprague-Dawley (Crl:CD®(SD)Br) rats were obtained from Charles River Breeding Laboratories, Inc., Newfield, NJ, USA
- Age at study initiation: 6 weeks of age
- Weight at study initiation: males, 195.8-229.8 g; females, 147.1-172.3 g
- Fasting period before study: no
- Housing: The F0 and Fl parental animals were housed individually in elevated stainless steel wire-mesh cages during the growth phase, and housed one male per one female in stainless steel breeding cages throughout the mating period. Following confirmation of mating or after a 3l-day mating period, the males were housed individually as they had been during maturation. On presumed gestation day 20, the mated F0 and F1 females were placed in individual polycarbonate nesting boxes for the remainder of gestation, and for the parturition and lactation periods. The females were returned to individual cages after weaning.
- Diet (e.g. ad libitum): basal diet (Purina Certified Rodent Chow 5002), offered ad libitum
- Water (e.g. ad libitum): tap water was offered ad libitum
- Acclimation period: 16 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 25 °C
- Humidity (%): 50 +/- 20%
- Air changes (per hr): not specified in the report
- Photoperiod (hrs dark / hrs light): 12 hrs/ 12 hrs

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
The amount of test material needed for each dose level was weighed and each level was premixed with approximately 200 g of feed until a homogenous mix was achieved. Each premix was added to the required amount of feed (weighed on a kilogram balance) and mixed again.
Fresh diets were mixed biweekly and presented weekly; excess diets were stored at room temperature until used. A 5-gram reserve sample of the test article and a 200-gram sample of the control article were taken at initiation of compound administration and stored in the freezer. Additionally, throughout the study, samples of each mix were sent to the sponsor.
Details on mating procedure:
The animals of the parental generation (F0 and F1) were paired one to one for mating after at least 10 weeks of dietary treatment. Each male was cohabitated with one female from the same group. During mating, a daily vaginal examination of each female was performed to determine the presence of sperm or the presence of a copulatory plug and the stage of estrous. The day of observation of sperm or plug was designated as Day 0 of gestation. Following a maximum 21-day mating period, females with no evidence of mating and normal estrous cycles, were placed with proven males in the same group for 10 additional days until mating was detected. When mating was detected, male and female rats were separated. Mated females were placed in nesting boxes on day 20 of presumed gestation.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were conducted to determine homogeneity of the test diet on samples prior to initiation of treatment, and on samples from study weeks 13-14, 17-18 and 23-24. Test diets were prepared at the intended low-, mid- and high-dose concentrations. Samples from the top, middle and bottom of the mixture were collected from each level and analyzed. Homogeneity analyses were done on each batch size used. Analyses of the test material in
the diet were conducted to determine 14-day stability at room temperature on mixes obtained prior to treatment initiation and for 21-day stability during study conduct. Concentration analyses were performed on samples of control and test diets for the first four mixes and every other mix thereafter.
Duration of treatment / exposure:
Exposure period: 10 weeks premating and through mating (up to 31 days), gestation and lactation phases
Premating exposure period (males): 10 weeks
Premating exposure period (females): 10 weeks
Frequency of treatment:
daily
Details on study schedule:
This study was designed to evaluate the reproductive toxicity of triclosan when fed daily to male and female rats for two generations To this end parental (F0, F1) mating behaviour and fertility as well as gestation parturition and lactation were investigated in addition to offspring (F growth and development. The test material was mixed with diet at concentrations of 300, 1000, or 3000 ppm (Groups 2-4, respectively) The control group (Group 1) received the basal diet. Twenty-five rats per sex per group were assigned to the study. Breeding was initiated after a ten-week growth phase. Thirty F1 rats per sex per group were selected for breeding to produce the F2 generation The growth phase for the F1 animals ranged from approximately 12-14 weeks. All F2 pups were sacrificed following weaning.
Doses / concentrations
Remarks:
Doses / Concentrations:
300, 1000, 3000 ppm
Basis:
nominal in diet
No. of animals per sex per dose:
25/sex/group (F0 generation)
30/sex/group (F1 generation)
Control animals:
yes, concurrent no treatment
Details on study design:
This study was designed to evaluate the reproductive toxicity of triclosan when fed daily to male and female rats for two generations.
To this end parental (F0, F1) mating behaviour and fertility as well as gestation parturition and lactation were investigated in addition to offspring (F growth and development.
The test material was mixed with diet at concentrations of 300, 1000, or 3000 ppm (Groups 2-4, respectively).
The control group (Group 1) received the basal diet.
Breeding was initiated after a ten-week growth phase.
Thirty F1 rats per sex per group were selected for breeding to produce the F2 generation.
The growth phase for the F1 animals ranged from approximately 12-14 weeks.
All F2 pups were sacrificed following weaning.
Body weight and food consumption data were collected at selected intervals throughout the study.
Reproductive and developmental toxicity was assessed by measuring duration of gestation, pregnancy rates, fertility, and pup viability growth, and development.

Examinations

Parental animals: Observations and examinations:
MORTALITY AND CLINICAL SYMPTOMS
The animals were observed twice daily for mortality and once for symptoms indicative of toxicity.

BODY WEIGHT
Body weight was recorded weekly during treatment and at terminal sacrifice for those males and females that were not confirmed mated.
Confirmed mated females were weighed on presumed gestation Days 0, 7, 14, and 20. Nursing dams were weighed on Days 0, 4, 7, 14, and 21 post partum.

FOOD CONSUMPTION AND COMPOUND INTAKE
Food consumption was calculated weekly during pre-mating in males and females, and for the females, on Days 7, 14, and 20 of gestation and days 4, 7, 14, and 21 of lactation. No food consumption data was obtained during the mating period.

REPRODUCTIVE TOXICITY
Reproductive toxicity was assessed by measuring duration of gestation, pregnancy rates, and fertility.

DELIVERY
The females were allowed to deliver their pups. They were checked for delivery four times daily (twice each in the morning and afternoon) for determination of the time of birth, and the presence of milk in each pup's stomach on the first day (day 0 after birth) was recorded.
Oestrous cyclicity (parental animals):
The estrous cycle was assessed daily during mating.
Sperm parameters (parental animals):
Sperm parameters were not assessed.
Litter observations:
PARAMETERS EXAMINED
On days 0, 4, 7, 14 and 21 after birth, the number of live and dead pups of each sex per litter, the body weight of each live pup, and clinical observations on live pups were recorded.

GROSS EXAMINATION OF DEAD PUPS
Gross necropsy on dead pups was done. The dead pups were examined grossly for cervical, thoracic, and abdominal visceral abnormalities and cause of death.

STANDARDISATION OF LITTERS
The litters were culled to 8 pups/litter on lactation day 4. In fact, all litters with more than eight pups were reduced to that number. Selection of pups for culling was by random card draw with equal numbers of males and females selected when possible. The culled pups were sacrificed by intraperitoneal injection and were also examined for abnormalities of the cervical, thoracic, and abdominal viscera prior to being discarded. The cause of death for pups found dead was recorded when evident.

WEANING AND SELECTON OF ANIMALS FOR F1 PARENTAL GENERATION
Each litter was weaned on day 21 of lactation. Cage side observations and pup counts were recorded daily for each litter. After all litters were weaned, one male and one female pup were selected by random card draw from the remaining pups of each litter (up to 2/sex/litter) to form the F1 parental generation. For litters without sufficient male or female pups, weanlings were randomly selected from the available pups of that sex to provide 30 male and 30 female weanlings per group. Records of the derivation of each pup were maintained in order to avoid sibling mating in the second generation.
After selection of the F1 parental generation, the remaining F1 pups were sacrificed and subjected to a gross examination of cervical, thoracic, and abdominal viscera for macroscopic abnormalities prior to being discarded.

F2 PUPS
Each litter was weaned on day 21 of lactation. Following weaning, weekly food consumption, body weights, and clinical observations were recorded. The animals were sacrificed at approximately 10 to 13 weeks of age and subjected to gross pathological examination as already described.
Postmortem examinations (parental animals):
Following delivery of the F1 pups, the F0 males were sacrificed; following weaning of the F1 pups, the F0 females were sacrificed also. The sacrifices were performed by exsanguination under sodium pentobarbital anesthesia, and the animals were subjected to a complete gross necropsy. The rats were not fasted prior to terminal sacrifice. The following organs and tissues were preserved in 10% neutral buffered formalin for possible future histopathological examination: vagina, uterus, ovaries, testes, epididymes, seminal vesicles, prostate, pituitary, liver, and all gross lesions.

Prior to fixation, the uterus of each female not delivering within 26 days of mating was examined for evidence of pregnancy and, if no evidence was found, ammonium sulfide staining was used to detect very early implantation scars. Each uterus of a female found dead or sacrificed in extremis was examined for implantations and the ovaries were examined for corpora lutea. When possible, the extent of development of implantations was determined and the gravid uterus was evaluated for resorptions, dead, or normally developing fetuses.
The sacrifice and necropsy procedures for the F1 parental animals were similar to the procedures for the F0 animals; although only for the F1 females were the number of implantation sites recorded at necropsy.
Statistics:
The control group values representing F0 and F1 parental body weights and food consumption of the premating phase (weeks 0-10 for the F0 generation and weeks 0-12 for the F1 generation), maternal body weights and body weight changes during gestation, maternal body weights and body weight
changes during lactation, and maternal food consumption during gestation and lactation were compared statistically to the data of the treated groups of the same sex and the statistical assessment was based on Levene’s test of homogeneity of variances, including ANOVA, the Terpstra-Jonchkeere nonparametric test for trend and Dunnett’s control vs treatment comparison where necessary.
The statistical analyses of F1 and F2 pup body weight data were based on Levene’s test of homogeneity of variances, including ANOVA, Dunnett’s control vs treatment comparison and the F-test, using the number of pups in each litter as the covariate. On the first day of lactation, the number of pups included both live and dead animals; on all subsequent days of lactation, only the live pup count was used. Tests for homogeneity of variances and ANOVA were evaluated at the 5% one-tailed probability level. When variances of untransformed data were heterogeneous, analyses were performed on transformed data to achieve variance homogeneity. When transformations were not successful in achieving homogeneity, analyses were performed on rank-transformed data. Control vs. compound-treated group mean comparisons of the above data were evaluated at the 5% two-tailed level; however, in the case of a significant trend, the data were evaluated at the 5% one-tailed level.
Reproductive indices:
The female fertility and male fertility indices were determined, as well as the gestation index.

Results and discussion

Results: P0 (first parental generation)

Details on results (P0)

SYSTEMIC TOXICITY DATA
There was no evidence of treatment related deaths in the parental generation. Clinical and cageside observations of the F0 and F1 animals during growth, gestation, and lactation did not indicate a compound-related effect.
During the F0 growth phase the mean body weight values occasionally were below control levels in the 3000 ppm group; however, the mean gain values did not indicate a treatment-related effect.
The F0 female mean body weight and food consumption values during gestation also did not indicate a treatment-related effect.
During lactation the mean mean body weight values at 3000 ppm were less than the corresponding control values, however, the mean weight gains were similar to the control gains. Therefore, no clear evidence of toxicity was indicated in clinical observation data, mean body weight values and food consumption values for the F0 animals during any phase.
The F1 mean body weights during the growth phase were below control values in both Groups 300 and 3000 ppm, but generally similar to control values in the 1000 ppm group. The mean body weight change values for the growth phase were generally similar each week, but a significant negative trend was displayed for the females for total mean changes (Weeks 0-11). This pattern also was evident for the males but not statistically significant. In both cases, the values of the 1000 ppm group were similar to the control values. Again, no evidence of a treatment-related effect was seen in the clinical observation and mean food consumption data. Mean F1 body weight values (Day 0, 7, 14, 20) and body weight change values (0-7, 14-20, 0-20) during gestation were significantly below the control values in the 3000 ppm group. This was also seen in 300 and 1000 ppm groups but only for the Day 20 weight. In the 3000 ppm group, mean lactation body weight values were also below the control levels, but the mean weight gain values did not indicate a treatment-related effect.

REPRODUCTION PARAMETERS
In F0 females, there was no indication of treatment influencing oestrous cycling, pregnancy rate, and gestation length. The gestation index was 100% for all groups.
F1 pregnancy rates did not indicate a dose-related effect. No influence of treatment on normal cycling behaviour of the F1 females was indicated. Length of gestation was similar for all groups

NECROPSY FINDINGS
In the F0 generation, the incidence of liver findings was slightly increased in the males (1000 and 3000 ppm) but not females, when compared to their respective control groups. These findings mainly involved discoloration, for further details, see the table below. Changes in the testes (size, texture and color) were noted in 2, 3 and 3 males in the 300, 1000 and 3000 ppm group, respectively. There were no other remarkable differences between the control and treated groups.
In the F1 generation, dilated pelvis(es) of the kidney were noted in 3, 1, 4 and 5 males in the control, the 300 ppm, the 1000 ppm and the 3000 ppm group, respectively, as well as in 4, 1, 4 and 6 females, respectively. Other gross pathology observations appeared sporadically.

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
(systemic parental toxicity)
Effect level:
3 000 ppm
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: no evidence of treatment related deaths or symptoms of toxicity in the parental generation (F0 and F1).
Key result
Dose descriptor:
NOAEL
Remarks:
(reproduction)
Effect level:
3 000 ppm
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: no adverse effects on reproduction in the parental generation (F0 and F1)

Results: F1 generation

Details on results (F1)

F1 PUPS
The F1 viability index (survival for das 0-4) was similar for Groups 0, 300 and 1000 ppm, but substantially lower for the 3000 ppm pups. The weaning index (survival for days 4-21), however, was similar for all groups. An apparent high-dose effect in the F1 pups was evidenced by a significant reduction in the mean adjusted body weights of both sexes during lactation.
At necropsy effects on the kidneys were reported for the F1 weanling of the 1000 and 3000 ppm groups. In fact, dilated kidneys and/or pe1vis(es) were observed in 7, 4, 9, and 14 pups in the control, the 300, the 1000 and the 3000 ppm group, respectively. Other findings included a missing tail and missing digits of the paw, and small testis, which occurred sporadically and were not considered compound-related. No gross lesions were observed in the
pups that died during lactation.
F2 PUPS
Survival of the F2 pups to weaning was slightly lower in the 3000 ppm compared to the control group. Although in the 3000 ppm group mean pup weight values were significantly below the control value at birth, they were above or similar to the control values at all other times.
Necropsy of the F2 pups found dead or sacrificed by lactation day 21 revealed dilated pelvis(es) in 4, 5, 3 and 2 pups in the control, the 300 ppm, the 1000 ppm and the 3000 ppm group, respectively, as most common finding. Further findings were sporadic and not related to treatment.
Necropsy of the F2 offsprings sacrificed after a growth phase again revealed dilated pelvis(es) in 9, 10, 15, and 8 pups from control, the 300 ppm, the 1000 ppm and the 3000 ppm group, respectively, as most common finding. Further findings were seen in liver, uterus, lungs, testes, and gastrointestinal tract; these findings appeared sporadically and did not follow a dose-related pattern.

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Remarks:
(neonatal, developmental toxicity)
Generation:
other: F1 and F2 pups
Effect level:
1 000 ppm
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: evidence of neonatal toxicity at 3000 ppm indicated by decreased survival of the F1 pups and suggested for the F2 pups. evidence of toxicity in the F1 pups indicated by effects on body weight at 3000 ppm during lactation.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Comparative summary of treatment-related findings:

Parameter

Genera­tion

Controls

300 ppm

1000 ppm

3000 ppm

Dose-response

Males

Females

Males

Females

Males

Females

Males

Females

Males

Females

Body weight

g

 

average weight on gestation Day 14

F0dams

/

348.4

/

338.6

/

356.0

/

343.8

/

F1dams

/

357.1

/

339.1

/

344.7

/

316.7*

/

+

average pup weight on lactation Day 21

F1pups

44.8

42.8

44.6

41.8

46.3

43.5

40.2*

37.7*

+

+

F2pups

40.7

39.5

41.0

39.3

44.1

41.2

38.5

36.9

Reproductive Performance

Viability index

%

F1pups

90

94

96

82*

+

F2pups

87

97

90

84

*statistically significant different from control p =<0.05

Main findings in the liver of F0 male animals at necropsy

Main findings in the liver of F0 male animals at necropsy

Test group

Control (0 ppm)

300 ppm

1000 ppm

3000 ppm

Number of animals examined

25

25

25

25

Findings in the liver

Thickened lobe

0

0

1

0

Dark

0

0

1

0

Pale area

0

0

0

1

Dark area

0

1

0

1

Raised area

2

0

1

0

Irregular shape

0

1

2

1

Enlarged

0

0

0

1

Pale

0

0

0

1

No such findings could be evidenced in the females

Applicant's summary and conclusion