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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
two-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13 February 2006 - 12 April 2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study, GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report Date:
2008

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Product name: TL-10-ST
Appearance: viscous liquid, straw light yellow colour

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, Germany
- Age at study initiation: 7-8 weeks (DRF study), 3-5 weeks (main study)
- Variation initial body weight: not above +-20% of mean weight for each sex
- Housing: macrolon cages
- Diet: ad libitum (Rat & Mouse No.3 Breeding Diet)
- Water: ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 40-70, upper values exceeded at several occasions for short periods (cleaning, meteorological circumstances)
- Air changes (per hr): about 10
- Photoperiod (hrs dark / hrs light): 12h/12h

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
The concentration of the test substance in the diets was adjusted during the different periods of the study to maintain the dose rates in the face of differing food consumption rates.
- The actual intake for males was 28.9, 85.8 and 261.9 mg/kg bw/day and for females 33.2, 97.1 and 302.3 mg/kg bw per day
Details on mating procedure:
- Premating period: at least 10 weeks, groups of 3 or 4/sex
- M/F ratio per cage: 1 male, 1 female
- Length of cohabitation: 2 weeks or until pregnancy occurred
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged: individually
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- Stability, homogeneity and content of the test substance in the diet were analysed using gas chromatography with flame ionisation detection (GC-FID) after extraction of the diet with a suitable solvent
- Test substance was distributed homogeneously in the first batches of diets prepared for the main study as demostrated by analysis of five samples taken at different locations in the food container of each diet. In four other batches the content of the test substance was checked by analysing one sample per concentration.
- The test substance was demonstrated to be stable in diet, stored at room temperature for 7 days, stored at 2-10 degrees C for 5 weeks and when stored at <-18 degrees C for 7 weeks
- Content of the test substance in all diets prepared was close to the intended concentrations with some minor exceptions
Duration of treatment / exposure:
After allocation to the treatment groups, animals were fed diets containing test substance from the start of the study, during the premating period of at least 10 weeks, during mating, gestation and lactation until sacrifice.
Frequency of treatment:
Continuously via 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 on PND 21
- Age at mating of the mated animals in the study: appr. 10 weeks
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
30 mg/kg bw/day
Basis:
nominal in diet
F0 and F1 gen. 250-500 mg/kg diet
Remarks:
Doses / Concentrations:
100 mg/kg bw/day
Basis:
nominal in diet
F0 anf F1 gen. 750-1500 mg/kg diet
Remarks:
Doses / Concentrations:
300 mg/kg bw/day
Basis:
nominal in diet
F0 anf F1 gen: 2250 - 4500 mg/kg diet
No. of animals per sex per dose:
F0: 28 male and 28 female rats per group (1 control and 3 dose groups)
F1: 28 male and 28 female rats selected in each group to rear next generation
Control animals:
yes, concurrent no treatment
Details on study design:
Dose levels of the main study were selected based on the results of a 1-generation dose-range finding study
The concentration of the test substance in the diets was adjusted during the different periods of the study to maintain the dose rates in the face of differing food consumption rates.
- The actual intake for males was 28.9, 85.8 and 261.9 mg/kg bw/day and for females 33.2, 97.1 and 302.3 mg/kg bw per day

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: checked daily in morning, additionally in afternoon for dead or moribund animals (on working days)

BODY WEIGHT: Yes
- Time schedule main study: males shortly before start of administration of the test substance and weekly afterwards. Females: shortly before the start of the administration of the test substance, once per week during mating, mated females on day 7, 14, 21 of gestation and on day 1,4, 7, 14, 21 of lactation. All animals were weighed on their scheduled necropsy date. After weaning body weights of F2 pups were recorded weekly.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption measured per cage by weighing the feeders
- Males: measurements approx. once a week, except during mating period
- Females: measurements approx. once a week during premating period and pregnancy and on day 1, 4 ,7 , 14 and 21 of lactation
- Calculation test substance intake: calculated from nominal dietary concentration of test substance, food consumption and mean body weight
Oestrous cyclicity (parental animals):
Daily for about 3 weeks prior to mating
Sperm parameters (parental animals):
- Epididymal sperm motility and reserve count for all males of all dose groups, count and morphology (high dose and control)
- Testicular sperm count (high dose and control), testis weight
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in [F1 / F2] offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies (days 1,4,7,14,21 of lactation), pup weight (day 1,4, 7, 14, 21 of lactation, weight gain. Number of runts defined as less than mean pup weight of the control group minus 2 standard deviations was calculated.

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities. Gross necropsy also on pups of dams that died during the lactation period and that showed external abnormalities at weaning.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals, after 77, 78 or 79 (F0) and 91, 92, 93 (F1) days of exposure
- Female animals: sperm-positive but not pregnant females 24 (F0) and 34 (F1) days after copulation; females without evidence of copulation 14 (F0) and 21 (F1) days after last day of mating period; dams on PN 21 (allowed to raise one litter per generation)
- PN 4 litters of more than 8 pups were adjusted by sacrificing the extra pups by random selection to yield as nearly as possible 4 males and 4 females per litter.
-After weaning selection of the animals for the next generation (28 males and 28 females) on PN21 the remaining animals were sacrificed.
GROSS NECROPSY: all parental animals
HISTOPATHOLOGY AND ORGAN WEIGHTS:
All parental animals: organ weights:adrenals, brain, kidneys, liver, pituitary gland, spleen, thyroid, organs and tissues that showed macroscopic alterations.
-Females: ovaries, uterus
-Males: epididymides, prostate, seminal vescles and coagulating glands, testes
Histopathological examination in all dose groups: liver and thyroid, control and high dose group: adrenals, epididymes, ovaries, pituitary gland, prostate, seminal vesicles and coagulating gland, spleen, testes, uterus, vagina, all macroscopic changes.
- Reproductive organs of males that failed to sire and females that were not mated or non-pregnant of the low and mid dose groups were microscopically examined.

Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring not selected as parental animals (one male and one female of each litter) and all F2 offspring were sacrificed at 21 days of age.
- These animals were subjected to postmortem examinations

GROSS NECROPSY:
- The F1 offspring not selected as parental animals (one male and one female of each litter) and all F2 offspring were sacrificed at 21 days of age.
All stillborn pups, pups found dead and pups that were terminated in a moribund condition during the study were examined for structural anomalies
and pathological changes. Pups of dams that died during lactation, pups that showed externmal abnormalities at weaning.
HISTOPATHOLOGY AND ORGAN WEIGHTS:
- The F1 offspring not selected as parental animals (one male and one female of each litter) and all F2 offspring were sacrificed at 21 days of age examined for skeletal anomalies
- At weaning 1 male and 1 female pup per litter subject to necropsy and gross pathological changes.
HISTOPATHOLOGY AND ORGAN WEIGHTS:
Pups necropsied: Organ weights: brain, spleen, thymus. Histopathology: organs and tissues with macroscopic abnormalities.
Carcasses of pups necropsied: examined for skeletal abnormalities of the ribs and sternum.
-
-All parental animals: organ weights:adrenals, brain, kidneys, liver, pituitary gland, spleen, thyroid, organs and tissues that showed macroscopic alterations.
-Females: ovaries, uterus
-Males: epididymides, prostate, seminal vescles and coagulating glands, testes
Histopathological examination in all dose groups: liver and thyroid, control and high dose group: adrenals, epididymes, ovaries, pituitary gland, prostate, seminal vesicles and coagulating gland, spleen, testes, uterus, vagina, all macroscopic changes.
- Reproductive organs of males that failed to sire and females that were not mated or non-pregnant of the low and mid dose groups were microscopically examined.
Statistics:
Clinical findings, number of mated and pregnant females, females with live pups: Fisher's exact probability test
Body weights, body weigth gains, organ weights, food consumption: one way analysis of ANOVA followed by Dunnetts multiple comparison test.
Number of implantation sites, live and dead pups: Kruskall-Wallis non-parametric analysis of variance followed by Mann-Whitney U-test.
Estrous cylicity: Fisher's exact test (number of acyclic animals and number of animals with prolonged estrous period), Kruskall-Wallis non-parametric analysis of variance followed by Mann-Whitney U-test for length of longest cycle, ANOVA followed by Dunnetts multiple comparisons tes for number of cycles per animal.
Sperm parameters: ANOVA followed by Dunnetts mutliple comparison test: epididymal and testicular sperm count, numerical sperm motility parameters. Kruskal-Wallis non parametric ANOVA followed by Mann-Whitney U-test for motility parameters expressed as percentage and sperm morphology.
Histopathological changes, anogenital distance, parameters for sexual maturation: Fisher's exact probability test.
Reproductive indices:
- pre-coital time
- duration of gestation
- mating index
- male fertility index
- female fertility index
- female fecundity index
- gestation index
- number of lost implantations
- post-implantation loss
Offspring viability indices:
- live birth index
- pup mortality day
- sex ratio day

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
no effects observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Other effects:
effects observed, treatment-related

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)
No mortality and no treatment related clinical signs.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
F0 generation
Mean Body weight males:
High dose group: statisitcally significantly decreased compared to controls in weeks 4 and 10
Mid dose group: statisitcally significantly decreased compared to controls in weeks 4, 8, 9, 10.

Mean body weigths females: Not statistically different from controls in all dose groups during premating, gestation and lactation

Mean body weight changes
males:
Statistically significantly decreased compared to controls all dose groups: week 3-4, mid dose group: additionally week 2-3.
females:
premating
high dose group: Statistically significantly decreased compared to controls week 0-1
gestation:
Only mid dose group: Statistically significantly decreased compared to controls GD14-21 in parallel with a decreased food consumption
lactation:
Mid dose group: Statistically significantly increased compared to controls days 1-4

F1 generation
Mean Body weight
males: Comparable to control group in all treatment groups
females:
premating:
Mid dose group: Statistically significantly decreased compared to controls weeks 2,3,6
gestation:
Mid and high dose group: Statistically significantly decreased compared to controls GD 21
lactation: no treatment related changes

Mean body weight changes
males:
high dose group: Statistically significantly decreased compared to controls in week 1-2
mid dose group: Statistically significantly decreased compared to controls in week 10-11

females
premating:
All dose groups: Statistically significantly decreased compared to controls week 1-2
gestation:
All dose groups: Statistically significantly decreased compared to controls GD14-21
lactation:
High dose group: Statistically significantly increased compared to controls days 14-21

Mean Food consumption
F0
Males
low and mid dose group:
Mean food consumption statistically significantly decreased compared to controls, week 4-6
Females:
premating
High dose group
Mean food consumption statistically significantly increased compared to controls: week 1-2
gestation:
Mid dose group: Mean food consumption statistically significantly decreased compared to controls GD 14-21
lactation:
Mid dose: Mean food consumption statistically significantly decreased compared to controls: days 1-7

F1
Males: comparable to controls in all dose groups
Females
premating:
High dose group: Mean food consumption statistically significantly increased compared to controls: week 1-2
gestation:
comparable to controls in all dose groups
lactation:
High dose group: Mean food consumption statistically significantly decreased compared to controls day 14-21

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS) FO
Test substance intake was calculated over the premating period (males and females), gestation period (females) and lactation period day 1-14 (females). As pups start eating considerable amounts during weeks 14-21 this period was not included in the calculation.
Test substance uptake in mg/kg bw /day
F0 males:
low dose: 26.8 (24.0-32.1)
mid dose: 81.2 (72.2-94.7)
high dose: 249.0 (223.9-294.6)
F0 females:
low dose premating: 29.8 (26.9-34.8) Gestation: 29.1 (22.5-32.4) lactation: 39.2 (36.7-41.7)
mid dose premating: 87.1 (78.2-100.1) Gestation: 82.1 (58.3-95.4) lactation: 109.2 (101.5-116.9)
high dose premating: 283.9 (253.4-321.8) Gestation: 265.2 (217.6 - 292.0) lactation: 357.7 (325.9 -389.5)
Overall intake males:
low dose: 28.9 mid dose: 85.8 high dose: 261.9
Overall intake females:
low dose: 33.2 mid dose: 97.1 high dose: 302.3

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS) F1
Test substance uptake in mg/kg bw /day
F0 males:
low dose: 31.0 (27.7-38.1)
mid dose: 90.5 (78.7-111.4)
high dose: 274.8 (245.9-335.9)
F0 females:
low dose premating: 30.9 (27.2-34.8) Gestation: 29.5 (24.8-32.4) lactation: 40.9 (38.1-43.7)
mid dose premating: 92.4 (80.8-102.6) Gestation: 90.0 (79.1-95.6) lactation: 121.3 (113.5-129.1)
high dose premating: 287.1 (255.6-334.1) Gestation: 268.3 (234.1 - 287.8) lactation: 351.8 (320.3 -383.3)
Overall intake males:
low dose: 28.9 mid dose: 85.8 high dose: 261.9
Overall intake females:
low dose: 33.2 mid dose: 97.1 high dose: 302.3

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
F0 generation
No treatment related differences from control
F1 generation
High dose group: the mean length of the longest cycle (5.0 days) was statistically singificantly increased compared to the controls (4.2 days). As a result also the mean number of cylces per animal was decreased (3.6 versus 4.0 in control). The effects on the cycle length was not considered of biological significance as the mean cycle length of 5 days is normal for this strain of rat and within the historical control data of the institute and the significant difference was also due to the low mean cycle length of the control group. In addtition the effect was not consistent across generations and microscopical examination of the reproductive organs did not reveal any adverse effect.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
F0 and F1 generation
No treatment related differences from control

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
F0 generation
No treatment related differences in pre-coital time, mating index, female fecundity index, male and female fertility index, duration of gestation, post implantation loss. All dams survived delivery and there were no stillborn pups.
F1 generation
No treatment related differences in pre-coital time, mating index, female fecundity index, male and female fertility index, duration of gestation, post implantation loss. All dams except one of the mid dose group survived delivery. Apart from the litters of this dam there were no stillborn pups in any of the groups. Three other dams of the mid dose group lost their litter between PN1 and PN 4. These effects were not considered toxicologically significant. The mean number of pups delivered was comparable between the groups.

ORGAN WEIGHTS (PARENTAL ANIMALS)
F0 generation
Absolute and relative liver and thyroid weights were increased compared to controls in male and female animals of the high dose group, relative liver and thyroid weights also in males of the mid dose group. Absolute and relative spleen weights were decreased compared to controls in high dose male and female animals. Absolute, but not relative spleen weights were also decreased in mid dose males.
F1 generation
Absolute and relative liver and thyroid weights were increased compared to controls in male and female animals of the high dose group, relative liverand absolute and relative thyroid weights were also increased in males of the mid dose group. Absolute and relative spleen weights were decreased compared to controls in high dose male and female animals. Absolute kidney weights were reduced compared to controls in low, mid and high dose males, absolute pituitary weights were decreased in mid and high dose males and absolute brain weights were reduced in mid dose male and female animals only.

GROSS PATHOLOGY (PARENTAL ANIMALS)
F0 generation
No treatment related changes were observed.
F1 generation
One female of the mid dose group was found dead with 8 newborn pups. Necropsy revealed increased transparent liquid in the thorax and one dead pup in the uterus. The cause of death could not clearly be established, but the findings are indicative of dystocia. Since this finding was seen in one female only it is not considered treatment related.

HISTOPATHOLOGY (PARENTAL ANIMALS)
F0 generation:
Microscopic observations did not reveal any treatment related findings in the reproductive organs including the pituitary. All 10 males examined and 8/10 females of the high dose group revealed the following changes in the liver: enlargement of periportal hepatocytes with increased eosinophilic cytoplasm indicative of a slight to moderate hypertrophy. This finding was not observed in the mid and low dose group animals. In the thyroid diffuse hypertrophy of follicular epithelial cells and reduction of colloid was observed in 3/10 low dose, 6-10 mid dose and 10/10 high dose males as well as in 5/10 high dose females. The finding reached statistical significance in the mid dose group males. No other treatment related microsopic findings were observed.

F1 generation
Microscopic findings did not reveal any treatment related findings in the reproductive organs including the pituitary. No other treatment related microsopic findings were observed.


Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Effect level:
29 mg/kg bw/day
Sex:
male
Basis for effect level:
other: There were no effects on the male or female reproductive systems in this study, up to the highest doses tested.
Dose descriptor:
NOAEL
Effect level:
97 mg/kg bw/day
Sex:
female
Basis for effect level:
other: There were no effects on the male or female reproductive systems in this study, up to the highest doses tested.
Dose descriptor:
NOAEL
Remarks:
Fertility
Effect level:
> 262 mg/kg bw/day
Sex:
male
Basis for effect level:
other: There were no effects on the male or female reproductive systems in this study, up to the highest doses tested.
Remarks on result:
other: Generation: P and F1 (migrated information)
Dose descriptor:
NOEL
Remarks:
Fertility
Effect level:
> 302 mg/kg bw/day
Sex:
female
Basis for effect level:
other: There were no effects on the male or female reproductive systems in this study, up to the highest doses tested.
Remarks on result:
other: Generation: P and F1 (migrated information)
Dose descriptor:
NOAEL
Remarks:
developmental toxicity
Effect level:
> 97 mg/kg bw/day
Sex:
female
Basis for effect level:
other: see 'Remark'
Remarks on result:
other: Generation: P and F1 (migrated information)
Dose descriptor:
NOAEL
Effect level:
86 mg/kg bw/day
Sex:
male
Basis for effect level:
other: According to the test report based on direct toxicity to the offspring at later stages of lactation. Other effects were not considered biologically relevant and/or test substance related.
Remarks on result:
other: Generation: F1 + F2 (migrated information)
Dose descriptor:
NOAEL
Effect level:
97 mg/kg bw/day
Sex:
female
Basis for effect level:
other: According to the test report based on direct toxicity to the offspring at later stages of lactation. Other effects were not considered biologically relevant and/or test substance related.
Remarks on result:
other: Generation: F1 + F2 (migrated information)

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, treatment-related
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings:
effects observed, treatment-related

Details on results (F1)

VIABILITY (OFFSPRING)
F0 offspring (F1 generation)
The mean number of pups delivered was comparable between treated and control groups. In the mid dose group the number of liveborn pups was statisitcally significantly decreased compared to controls and the number of stillborn pups (due to only one dam with 11 of 12 pups stillborn) and the number of pups which were lost between PN1 and PN4 were statistically significantly increased. These effects are not considered toxicologically significant. No such effects were observed in the low and high dose groups. All pups of all groups remained alive after PN4. Sex ratio was comparable in all groups.

F1 offspring (F2 generation)
The mean number of pups delivered was comparable between treated and control groups. In the mid dose group the number of liveborn pups was statisitcally significantly decreased compared to controls and the number of stillborn pups was statistically significantly increased. On PN1 the number of pups alive was statistically significantly decreased in the mid dose group and the number of pups which were lost between PN1 and PN4 were statistically significantly increased. No such effects were observed in the low and high dose groups. After PN 4 only 3 pups of the control group and 2 of the high dose group died until weaning on PN 21. On PN1 the sex ratio was statistically significantly decreased in the high dose group, but this is considered due to a high number of males and a low number of females in the control group rather than a treatment related effect.

CLINICAL SIGNS (OFFSPRING)
F0 generation offspring (F1 generation)
In the F0 generation, the number of runts was statistically significantly increased (on a single animal basis, but not on a litter basis except for the mid dose group) in all dose groups on PN 1, and in the mid dose group also on PN 4, 7 and 14. There was no dose response relationship on the number of runts at any time point. Furthermore it is noted, that most of the runts in the low dose of the F0 generation are from the 2 dams with the lowest body weight of the group. As the increase in the number of runts in the low dose group PN 1 was not observed consistently across generations, it is not considered to be biologically significant. The increased numbers of runts in the treated pups in the mid and high groups in both generations on PN 1 could indicate systemic toxicity to the pups in utero. The number of cold pups and pups with no milk in stomach (5 pups of one litter) was statistically significantly increased in the F1 mid dose group on PN 1. No other effects were observed.
F1 generation offspring (F2 pups)
The number of runts was statistically singnificantly increased in the late lactation period on PN 14 and 21 in the low dose group and in the mid and high dose group during the entire lactation period. Statistical significance on a litter basis was only reached on PN 14 and 21. In the mid dose group the number of cold pups and pups with no milk in the stomach (5 pups in pone litter) was statistically significantly increased on PN 1.


BODY WEIGHT (OFFSPRING)
F0 generation offspring (F1 pups)
In the mid dose group mean pup weights were statistically significantly decreased on PN 1 and 7. However, when compared on a litter basis the effects on body weights of the pups on day 1 are not statistically significant. Pup weight changes in that group were comparable to controls during the entire lactation period. In the high dose group mean pup weights of the males were statistically significantly decreased on PN 7 and 21. PN 7 pup weights did not show a dose response. Pup weight changes of the high dose group were statistically significanly reduced between PN 14 and 21.

F1 generation offspring (F2 pups)
On a single animal basis pup weights in all treated groups were statistically significantly decreased during the entire lactation period, except for the mid dose group on PN 4. However between PN1 and 7 no clear dose response was observed. Pup weight changes were statistically significantly decreased between PN 7-21. From day 14 to 21 the weight reduction showed a dose response. In addition pup weight change in the high dose group was statistically significantly decreased when compared to the control between PN 1 and 4. However, when compared on a litter basis the effects on body weights of the pups on day 1 are not statistically significant.

SEXUAL MATURATION (OFFSPRING)
F1 offspring (F2 pups)
The anogenital distance of the female F2 pups of the mid and high dose groups was statistically significantly decreased when compared to controls. This finding was rather related to the lower body weight of this groups and not an indication of retarded sexual maturation.
No statistically significant difference between treated animals and controls was observed for vaginal opening. The day preputial separation was reached in the males of the high dose group was statistically significantly increased conmpared to controls (45.12 days versus 43 days). This finding was rather related to the lower body weight of this group from PN 28 to 42 and not an indication of retarded sexual maturation.

Effect levels (F1)

open allclose all
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
> 262 mg/kg bw/day
Sex:
female
Basis for effect level:
other: There were no effects on the male or female reproductive systems in this study, up to the highest doses tested.
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
> 302 mg/kg bw/day
Sex:
male
Basis for effect level:
other: There were no effects on the male or female reproductive systems in this study, up to the highest doses tested.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Table 7.8.1.1 Summary of the significant absolute and relative organ weight changes in male parental animals in the 2-generation reproductive toxicity study

F0-generation

F1-generation

Low

Mid

High

Low

Mid

High

Organ weight

A

R

A

R

A

R

A

R

A

R

A

R

Brain

-

-

-

-

-

-

-

-

decrease

-

-

-

Kidneys

-

-

-

-

-

-

decrease

-

decrease

-

decrease

-

Liver

-

-

-

increase

increase

increase

-

-

-

increase

increase

increase

Pituitary

-

-

-

-

-

-

-

-

decrease

-

decrease

-

Seminal vesicles

decrease

decrease

-

-

-

-

-

-

-

-

-

-

Spleen

-

-

decrease

-

decrease

decrease

decrease

-

-

-

decrease

decrease

Thyroid

-

-

-

increase

increase

increase

-

-

increase

increase

increase

increase

A: absolute weight; R: relative weight

Table 7.8.1.2 Summary of the significant absolute and relative organ weight changes in parental females in the 2-generation reproductive toxicity study

F0-generation

F1-generation

Low

Mid

High

Low

Mid

High

Organ weight

A

R

A

R

A

R

A

R

A

R

A

R

Brain

-

-

-

-

-

-

-

-

decreased

-

-

-

Liver

-

-

-

-

increased

increased

-

-

-

-

increased

increased

Spleen

-

-

-

-

decreased

decreased

-

-

-

-

decreased

decreased

Thyroid

-

-

-

-

increased

increased

-

-

-

-

increased

increased

A: absolute weight; R: relative weight

Table 7.8.1.3 Mean terminal body weights and significant organ weights for males of F0 and F1 generations

Dose Group

Organ

Generation

0

Low

Mid

High

Mean terminal body weight

F0

405.1

390.7

386.2

389.5

F1

399.5

386.8

383.5

381.3

Mean absolute organ weight (g)

Kidney

F0

2.338

2.244

2.231

2.288

F1

2.278

2.112**

2.110**

2.140*

Liver

F0

14.4

13.993

14.579

15.986**

F1

14.312

13.544

14.088

15.357*

Pituitary

F0

0.015

0.014

0.014

0.014

F1

0.016

0.015

0.014*

0.014*

Spleen

F0

0.744

0.704

0.681**

0.646***

F1

0.746

0.686*

0.692

0.646***

Thyroid

F0

0.023

0.022

0.025

0.029***

F1

0.023

0.024

0.026*

0.029***

Mean organ weights relative to terminal body weight (g/kg bw)

Kidney

F0

5.786

5.750

5.780

5.875

F1

5.601

5.468

5.520

5.616

Liver

F0

35.458

35.783

37.740**

41.018***

F1

35.184

35.006

36.760*

40.240***

Pituitary

F0

0.036

0.035

0.036

0.036

F1

0.040

0.039

0.038

0.038

Spleen

F0

1.849

1.805

1.766

1.662***

F1

1.835

1.775

1.810

1.691**

Thyroid

F0

0.056

0.058

0.065**

0.075***

F1

0.057

0.062

0.068**

0.075***

*/**/*** statistically significantly different to the control group p 0.05/ 0.01/ 0.001

Table 7.8.1.4 Mean terminal body weights and significant organ weights for females of F0 and F1 generations

Dose Group

Organ

Generation

0

Low

Mid

High

Mean terminal body weight

F0

266.4

264.2

264.1

265.5

F1

262.1

259.3

254.0

259.5

Mean absolute organ weight (g)

Liver

F0

13.318

13.348

12.798

16.002***

F1

13.137

13.230

12.742

15.974***

Spleen

F0

0.506

0.494

0.470

0.452**

F1

0.507

0.508

0.486

0.446**

Thyroid

F0

0.020

0.019

0.021

0.023***

F1

0.020

0.020

0.020

0.023***

Mean organ weights relative to terminal body weight (g/kg bw)

Liver

F0

49.938

50.501

48.488

60.131***

F1

50.059

51.105

50.107

61.521***

Spleen

F0

1.902

1.872

1.783

1.705**

F1

1.937

1.963

1.914

1.718***

Thyroid

F0

0.074

0.074

0.080

0.086***

F1

0.076

0.079

0.079

0.090***

**/*** statistically significantly different to the control group p 0.01/ 0.001

Table 7.8.1.5 Delivery, pup and litter data for F0 and F1 generations

    Dose group    
 Effect  0  low  mid  high
 F0        
 Mean No pups delivered  10.72  9.79  9.92  10.71
  No of liveborn  265  233  236*  249
No of stillborn    3   2   12   8
 No of pups lost (dying, missing/cannibalized)        
  Days 1 -4  28  29  60***  24
  Days 5 -7  0  0  0  0

                Days 8-14

0

0

0

0

                Days 15-21

0

0

0

0

Mean no. live pups/litter (PN1)

10.6

9.71

9.44

10.38

Sex ratio on PN1 (M/F)

132/136

104/131

125/123

134/123

No. pups alive Day 21

184

171

147

178

F1:

Mean  no. of pups delivered

10.36

10.50

9.86

9.58

                No. of liveborn

258

250

251***

249

                No. of stillborn

1

2

25***

0

No. of pups lost (dying, missing and/ or cannibalized) on:

                Days 1-4

7

1

23**

5

                Days 5-7

2

0

0

2

                Days 8-14

0

0

0

0

                Days 15-21

0

0

0

0

Mean no. live pups/litter (PN1)

10.32

10.42

9.3*

9.58

Sex ratio on PN1 (M/F)

145/114

136/116

132/144

110**/1392

No. pups alive Day 21

191

191

188

203

         

*/**/*** statistically significantly different to the control group p 0.05/ 0.01/ 0.001. 1 due to 1 animal with 11 of 12 pups stillborn

2 statsitcal significance due to high number of males and low number of females in the control. No biological significance

7.1.8.6 Clinical observations in pups of F0 and F1 generations on Days 1-21 of lactation

Dose Group

0

Low

Mid

High

F0

Runts

                Day 1

4(2)

19***(7)

110***(16)**

31***(9)

                Day 4

1

3(3)

21***(8)**

4(4)

                Day 7

2(2)

1

21***(7)

4(4)

                Day 14

2(2)

2(1)

16***(3)

3(3)

                Day 21

2(2)

0

6(3)

2(2)

F1

Runts

                Day 1

10(4)

18(6)

37***(8)

41***(7)

                Day 4

8(3)

12(5)

23**(4)

40***(7)

                Day 7

5(3)

12(5)

24***(7)

48***(9)

                Day 14

0

17***(7)**

13***(7)**

14***(11)***

                Day 21

4(3)

28***(11)*

41***(13)**

84***(16)***

*/**/*** statistically significantly different to the control group p 0.05/ 0.01/ 0.001

figures in brackets represent the number of litters with pups showing the observation.

Applicant's summary and conclusion

Conclusions:
In a two-generation reproductive toxicity study with the test substance, no treatment related differences were observed in pre-coital time, mating index, female fecundity index, male and female fertility index, duration of gestation and post-implantation loss. With the exception of one mid dose dam (C521) of the F1 generation, all dams delivered and there were no dams with stillborn pups. The mean number of pups delivered was comparable between the groups. There was no effect on sperm parameters at necropsy and there were no treatment related microscopic findings in the reproductive organs of either generation.
No effects on male or female reproductive system were observed up to the highest dose, and therefore, the NOAEL is greater than approx. 262 and 302 mg/kg bw/day for male and female animals, respectively.
The low dose of 29 mg/kg/day is considered to be the NOAEL for parental toxicity in males. This is based on thyroid weight changes in the mid and high dose males of both generations, and histopathological changes in this organ. The mid dose of approximately 97 mg/kg/day is considered the NOAEL for parental toxicity in females.
In a conservative approach the EU risk asssessment derived a NOAEL of 29 mg/kg bw/day for developmental toxicity. This was based on an increase in the number of runts on PND 1 and a decrease in mean pup weights obseved in the mid and high dose groups of both generations. However, as indicated in the original study report, the effects on PN1 are likely a statistical artefact of the study and not biologically relevant for the following reasons: In this particular study the body weights of the control animals were at the high end or even higher than recent historical control body weights of the test institute and are in a relatively narrow range with a comparatively low standard deviation. Therefore the statistical evaluation overestimates the effects identifying more animals as runts. When related to historical control data in the F0 generation all mean body weights of the low and high dose group are within the historical control range. Furthermore, when compared on a litter basis the effects on body weights of the pups on day 1 are not statistically significant in the F0 and F1 generation except for the F0 mid dose group. When looking at single animal data, runts on day 1 stem from mother animals with considerably lower body weights compared to the means of the respective groups and the effects on body weights are likely related to maternal toxicity. The increase in the number of runts as well as the decrease in pup weight in the high dose group towards the end of the lactation period is considered as toxicity to the offspring. Rather the reduced food intake (reduced palatability) is considered to be the cause for the retarded growth during the last week of lactation. Therefore the original study report only considered reduced body weights and numbers of runts at the end of the lactation period in the high dose group as substance related effects in this study. These effects are due to direct substance intake of the pups via the feed and are not considered as developmental toxicity. Therefore it can be concluded that in this study the substance did not show developmental toxicity. Direct toxicity via substance intake of the pups from the diet was seen at the high dose and the NOAEL for direct toxicity to the offspring can be considered the mid dose of 97 mg/kg bw.
Executive summary:

In the 2-generation study groups of 28 male and female Wistar rats received daily dietary doses of

2,2-bis(chloromethyl)trimethylene bis(bis(2-chloroethyl)phosphate)

of approximately 0, 28.9, 85.8 and 261.9 mg/kg bw (males) and 0, 33.2, 97.1 and 302.3 mg/kg bw (females) over two successive generations.

The dose selection was based on a preliminary dose range finding study with the same species, strain and route of administration.

The animals were fed diets containing the test substance from the start of the study, during the premating period of at least 10 weeks, during gestation and lactation until sacrifice. The concentration of the test substance in the diets was adjusted during the different periods of the study to maintain the dose rates in the face of differing food consumption rates. Dams were allowed to raise one litter. At the end of the lactation period, pups were weaned and selected for the next generation or sacrificed. F0-and F1-dams were sacrificed at or shortly after weaning. F0- and F1- males were sacrificed after at least 11weeks of exposure (for sperm analyses and necropsy).

The following effects were considered as treatment related and consistent parental effects: increased relative thyroid weight in the F0- and F1-males of the mid-dose group, increased thyroid weight of the F0-and F1-animals of the high-dose group,increased liver weight of the F0- and F1-animals of the high-dose group and decreased spleen weight in the F0- and F1-animals of the high-dose groups.

Multifocal periportal hepatocellular hypertrophy in the liver of the F0-animals of the high-group was considered to be a treatment-related effect. Activated/diffuse hypertrophy was observed in the thyroid in 0, 3, 6 and 10 male animals of the control, low-, mid- and high dose, respectively. In the females this effect on the thyroid was only observed in the animals of the high-dose group. The effect on the thyroid in the male animals of the low-dose group did not reach the level of significance. No reproductive toxicity was observed in this study. There was no developmental toxicity, but systemic toxicity to the offspring in the high-dose groups due to direct intake of the test substance, decreased body weight and numbers of runts at the end of the lactation period in the high dose F1 and F2 pups and decreased relative spleen weight in F2-pups of the high dose group.

The NOAEL for parental toxicity of the test substance is 29 mg/kg bw/day for male rats and 97 mg/kg bw/day for female rats based on the histopathological changes in the liver and thyroid.

The NOAEL for toxicity to offspring and developmental toxicity of 29 mg/kg was derived in the EU risk assessment. This was based on an increase in the number of runts on PN 1 and a decrease in mean pup weights observed in the mid and high dose groups of both generations. However,

as indicated in the original study report, the effects on PN1 are likely a statistical artefact of the study and not biologically relevant for the following reasons: In this particular study the body weights of the control animals were at the high end or even higher than recent historical control body weights of the test institute and are in a relatively narrow range with a comparatively low standard deviation. Therefore the statistical evaluation overestimates the effects identifying more animals as runts. When related to historical control data in the F0 generation all mean body weights of the low and high dose group are within the historical control range. Furthermore, when compared on a litter basis the effects on body weights of the pups on day 1 are not statistically significant in the F0 and F1 generation except for the F0 mid dose group. When looking at single animal data, runts on day 1 stem from mother animals with considerably lower body weights compared to the means of the respective groups and the effects on body weights are likely related to maternal toxicity. The increase in the number of runts as well as the decrease in pup weight in the high dose group towards the end of the lactation period is considered as toxicity to the offspring. Rather the reduced food intake (reduced palatability) is considered to be the cause for the retarded growth during the last week of lactation. Therefore the original study report only considered reduced body weights and numbers of runts at the end of the lactation period in the high dose group as substance related effects in this study. These effects are due to direct substance intake of the pups via the feed and are not considered as developmental toxicity. Therefore it can be concluded that in this study the substance did not show developmental toxicity. Direct toxicity via substance intake of the pups from the diet was seen at the high dose and the NOAEL for direct toxicity to the offspring can be considered the mid dose of 97 mg/kg bw.

This study is conducted according to GLP and satisfies the guideline requirement for a 2 -generation reproductive study according to OECD Guideline No.416. The study is considered as reliable without restrictions.