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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
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

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: OECD guideline 416 2-generation study in rats
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
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:
Concentration of the test substance in the diets was adjusted to achieve a test substance constant intake
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
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
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 the diet
Duration of test:
- 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
Basis:
nominal in diet
F0 and F1 250 to 500 mg/kg diet
Remarks:
Doses / Concentrations:
100 mg/kg bw
Basis:
nominal in diet
F0 anf F1 750 to 1500 mg/kg diet
Remarks:
Doses / Concentrations:
300 mg/kg bw
Basis:
nominal in diet
F0 and F1 2250 to 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 selection rationale: based on preliminary 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

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily on saturdays, sundays, public holidays, twice daily on working days
DETAILED CLINICAL OBSERVATIONS: No
BODY WEIGHT: Yes
- Time schedule for examinations:
Body weights of male and female rats were recorded shortly before the
start of administration of the test substance and weekly thereafter during the premating
period.
Males were weighed once per week during the mating period until sacrifice. Females
were weighed once per week during mating and mated females were weighed on days
0, 7, 14 and 21 during presumed gestation and on day 1, 4, 7, 14 and 21 of lactation. In
addition, the animals were weighed on their scheduled necropsy date in order to
calculate the correct organ to body weight ratios.
After weaning the body weights of the F2-pups were recorded weekly.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes
Food consumption was measured per cage by weighing the feeders.
Food consumption of male rats was measured approx. weekly, except during the mating
period. Food consumption of female rats was measured approx. weekly during the
premating period. Food consumption of mated females was recorded weekly during
pregnancy and on day 1, 4, 7, 14 and 21 of lactation.
The results were expressed in g per animal per day and g per kg body weight per day.
The intake of the test substance per kg body weight per day was calculated from the
nominal dietary concentration of the test substance, the food consumption and the mean
body weight in the pertaining period.

- Time schedule for examinations:
Gross necropsy and histology of parental animals
All surviving male and female parent rats were euthanized by exsanguination from the
abdominal aorta under CO2/O2 anaesthesia and then examined grossly for pathological
changes.
Samples of the following tissues and organs of all parent animals were preserved in a
neutral aqueous phosphate-buffered 4% solution of formaldehyde except for the testes
which was preserved in Bouin's fixative:
- adrenals**
- brain
- epididymides *(left cauda which was used for sperm analysis (see section
4.10.12)
- kidneys**
- liver**
- ovaries*
- pituitary gland*
- prostate*
- seminal vesicles and coagulating glands*
- spleen**
- testes*(right testis was preserved in Bouin’s fixative, the left one was used for
sperm analysis
- thyroid**
- uterus* (after counting of the implantation sites2)
- vagina*
- organs and tissues that showed macroscopic abnormalities
The following organs were weighed (paired organs together) as soon as possible after
dissection to avoid drying: 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
Tissues marked with an asterisk (* or **) were embedded in paraffin wax, sectioned at
5 μm, and stained with haematoxylin and eosin, except for sections of the testes, which
were stained with PAS. Microscopic examination was performed on organs marked
with one asterisks of all rats of both generations of the control and high-dose groups and
with two asterisks of the first 10 male and 10 female animals of the F0-generation of
the control and high-dose groups. An effect was observed in the liver and thyroid of the
F0-animals of the high-dose groups. For that reason the micoscopic examinations were
extended to the low- and mid-dose groups of these organs.
In addition, reproductive organs of males that failed to sire (did not mate or mated
females were not pregnant) and females that were non-mated or non-pregnant, of the
low- and mid-dose groups, were microscopically examined.

Ovaries and uterine content:
number of lost implantations = number of implantations sites - number of pups born
alive
- post-implantation loss = [(number of implantation sites - number of pups born
alive)/number of implantation sites] x 100
Fetal examinations:
gestation index = (number of females with live pups/number of females
pregnant) x 100
- live birth index = (number of pups born alive/number of pups born) x 100
- pup mortality day n = (number of dead pups on day n/total number of pups
on day n) x 100
- sex ratio day n = (number of live male pups on day n/ number of live pups on day n) x
10

At weaning 1 male and 1 female pup per litter was subjected to a thorough necropsy.
Pups were euthanized by exsanguination from the abdominal aorta under CO2/O2
anaesthesia and then examined grossly for pathological changes. Special attention was
paid to the organs of the reproductive system.
The following organs were preserved in a neutral aqueous phosphate-buffered 4%
solution of formaldehyde for possible future examination:
- brain
- spleen
- thymus
- organs and tissues that showed macroscopic abnormalities
After necropsy, the carcasses of the male and female F1-pups selected for necropsy
were stored at <-18°C. After thawing, the carcasses were eviscerated and thereafter the
trunk of the pups was fixed in 70% alcohol, and then cleared in potassium hydroxide
and stained with Alizarin Red S modified after Dawson (1926). They were examined
for skeletal abnormalities of the ribs and sternum and then retained.
Statistics:
- Clinical findings were evaluated by Fisher's exact probability test.
- Body weight, body weight gain, organ weights and food consumption data were
subjected to one-way analysis of variance (ANOVA) followed by Dunnett's
multiple comparison test.
- Fisher's exact probability test was used to evaluate the number of mated and
pregnant females and females with live pups.
- Number of implantation sites, live and dead pups were evaluated by
Kruskal-Wallis nonparametric analysis of variance followed by the Mann Whitney
U-test.
- Estrus cyclicity was evaluated by Fisher’s exact test (number of acyclic animals
and number of animals with prolonged estrus period), ANOVA followed by Dunnett’s
multiple comparison test (number of cycles per animal) and Kruskal-Wallis non
parametric ANOVA followed by Mann-Whitney U test (length of the longest cycle).
- Sperm parameters were evaluated by ANOVA followed by Dunnett’s multiple
comparison test (epididymal and testicular sperm count and numerical sperm motility
parameters) or by Kruskal-Wallis non parametric ANOVA followed by Mann-
Whitney U test (motility parameters expressed as a percentage and sperm
morphology).
- Histopathological changes, anogenital distance and parameters for sexual maturation
were evaluated by Fisher's exact probability test.
Indices:
Selection relevant for developmental toxicity:
- female fertility index = (number of pregnant females/number of females
placed with males) x 100
- female fecundity index = (number of pregnant females/number of females mated) x
100
- gestation index = (number of females with live pups/number of females
pregnant) x 100
- live birth index = (number of pups born alive/number of pups born) x 100
- pup mortality day n = (number of dead pups on day n/total number of pups
on day n) x 100
- sex ratio day n = (number of live male pups on day n/ number of live pups on day n) x
100
- number of lost implantations = number of implantations sites - number of pups born
alive
- post-implantation loss = [(number of implantation sites - number of pups born
alive)/number of implantation sites] x 100
Historical control data:
Historical control data on mean pup weights on PND 1 and mean number of pups delivered were included in the report.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
No test substance related clincal signs were observed in F0 females. No effects on body weights were observed in F0 females.
Mean body weights in F1 females of the mid dose group were decreased in weeks 2, 3 and 6. Mean body weights in F1 females of the mid and high dose group were decreased on gestation day 21. Mean food consumption of the females of the mid and high dose groups was decreased during gestation and lactation.
High dose females of the F0 and F1 generation showed increased absolute and relative liver and thyroid weights and decreased absolute and relative spleen weights.
No treatment related macroscopic findings were reported in the F0 and F1 females.
Microscopic alterations included slight to moderate hepatocellular hypertrophy in 8/10 females of the high dose F0 group. In the thyroid diffuse hypertrophy of the follicular epithelial cells an a reduction of colloid were observed in 5/10 high dose F0 females, but this finding did not reach statistical significance in females.
The mid dose of 97 mg/kg bw is considered the NOAEL for maternal toxicity.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Effect level:
97 mg/kg bw/day (actual dose received)
Basis for effect level:
other: developmental toxicity
Dose descriptor:
NOAEL
Effect level:
302 mg/kg bw/day (actual dose received)
Basis for effect level:
other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
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 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 cosidered 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 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)
On a single animal basis 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)
On a single animal basis 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 bais 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 PN 1 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 (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.
Necropsy findings:
At necropsy of the pups one low dose F1 pup showed enlarged thymus. There were no other findings in either generation.
Organ weights:
Absolute spleen weight was decreased in high dose F0 pups and all treated F1 pups. Relative spleen weight was decreased in high dose F1 pups only. Absolute brain weight was increased in all treated F1 pups while relative weight was significantly increased. Absolute thymus weight of low and high dose F1 pups was also decreased, but relative thymus weights were comparable to controls. Except the change in relative spleen weight in the high dose F1 pups all organ weight changes are considered to be related to pup weight rather than an organ specific effect. The reduction in relative spleen weight in the high dose F1 pups is considered a direct effect of the test substance intake and not a developmental effect.
No skeletal abnormalities or retarded ossification was observed in the ribs of F1 pups.

Effect levels (fetuses)

open allclose all
Dose descriptor:
NOAEL
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
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.

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

Table 7.8.2.1 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 7.8.2.2 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 7.8.2.3 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/ 0.001.1due to 1 animal with 11 of 12 pups stillborn

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

7.8.2.4 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 in Wistar rats, that received doses of 30, 100 or 300 mg of the test substance per kg bw via the diet, no treatment related differences were observed in duration of gestation and post-implantation loss. With the exception of one mid dose dam 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. 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 assessment derived a NOAEL of 29 mg/kg bw/day for developmental toxicity. This was based on an increase in the number of runts on PND1 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.
Executive summary:

In a 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. 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. No skeletal abnormalities or retarded ossification was observed in the ribs of F1 pups.

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.

In a conservative approach the EU risk assessment (European Commission, 2008a) derived a NOAEL of 29-mg/kg bw/day for developmental toxicity. This was based on an increase in the number of runts on PN1 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.