Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
A discussion and report on the read across strategy is given as an attachment in Section 13.
Cross-referenceopen allclose all
Reason / purpose:
read-across: supporting information
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
A discussion and report on the read across strategy is given as an attachment in Section 13.
Reason / purpose:
read-across source
Reason / purpose:
reference to same study
Remarks:
Sections 7.5.1. and 7.8.2
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No test item related clinical signs wereobserved in males or females in any group. In group 2, one female (no. 64)had hunched posture and chromodacryorrhea on days 1 and 2 as well as ruffled fur from day 1 to 3 post partum. Due to isolated occurrence in the low-dose group, these observations were considered not to be related to the treatment with the test item.
Mortality:
mortality observed, treatment-related
Description (incidence):
With exception for one female in group 4, all animals survived until the scheduled necropsy. In group 4, body weight loss up to 24% was noted in one female (no. 85) from the beginning of treatment. This female was terminated for ethical reasons on day 12 of the pre-pairing period. No findings were noted during necropsy of this female. The early termination was considered to be a result of the treatment with the test item.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Males - Pre-Pairing, Pairing and Post-Pairing Periods
Treatment with the test item caused dose-dependent reduction in body weight and body weight gain in groups 3 and 4. Mean body weight was greater than 90% of the control group value throughout the treatment period. Mean body weight gain in order of ascending dose levels was 12.4%, 12.6%, 9.7% and 1.8% during the pre-pairing period, 8.2%, 7.5%, 8.1% and 8.4% during the pairing period, and 2.9%, 4.0%, 3.4% and 3.4% during the post-pairing period. In group 4, body weight loss up to 3.5% was noted on day 4 of the pre-pairing period followed by statistically significantly reduced body weight gain recorded until the end of this period. Body weight gain during the pairing and the post-pairing periods was similar to the control values (if expressed as a percentage of body weights in the group at the beginning of the respective period). Consequently, lower body weights compared to control were recorded from day 4 of the prepairing period, and which remained approximately 91-92% of the mean control group body weight from day 10 until the completion of the study. This reduction was statistically significant during the entire study period. In group 3, statistically significantly reduced body weight gain was noted from day 7 to 13 of the pre-pairing period and body weight gain similar to that recorded in the control group during the remaining study period. This resulted in slightly, not statistically significantly, lower body weights recorded from day 7 ofthe pre-pairing period onwards. In group 2, body weights and body weight gain weresimilar to the respective values in the control group.

Females - Pre-Pairing, Gestation and Lactation Periods
Treatment with the test item caused a reduction in body weights and body weight gain in group 4. Mean body weight gain in order of ascending dose levelswas 9.4%, 10.2%, 8.0% and 2.8% during the pre-pairing period, 55.1%, 48.6%, 49.8%and 43.8% during the gestation period, and 6.5%, 7.9%, 9.7% and 3.6% during the lactation period. Mean values ofbody weights and body weight gain were not calculated for females during the pairing period due to different length of this period for individual females. In group 4, body weight loss up to 5.8% was recorded on day 4 of the pre-pairing period and reduced body weight gain until the completion of the study. This reduction was statistically significant during the entire pre-pairing period and on days 14 and 20 of the gestation period. Consequently, reduced body weights were recordedfrom day 4 of the pre-pairing period until the completion of the study with statistical significance on the most ofthe days. Nevertheless, mean
bodyweight was no less than 94% ofthe control value throughout the pre-pairing period and the first week of the gestation period, but fell to 86% on lactation day 4. In groups 2 and 3, body weight and body weight gain were considered not to be affected by the treatment. In group 2 body weights and body weight gain were slightly higher than the respective control values during the entire study, with statistical significance on day 0 post coitum. The differences observed in this group were considered to be due to biological variability. In group 3, slightly, not statistically significantly lower body weight gain was noted during the pre-pairing and gestation periods. Body weights remained similar to the control values.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Males: Pre- and Post-Pairing Periods
Treatment with the test item caused a dose-dependent, reversible reduction in food consumption in groups 3 and 4. In order of ascending dose levels, mean food consumption was 22.3, 22.6, 21.3 and 16.5 g/animal/day during the pre-pairing period and 22.8, 23.2, 22.8 and 22.7 g/animal/day during the post-pairing period. In group 4, reduction in food consumption was statistically significant during the entire prepairing period. During the post-pairing period, food consumption in this group recovered and was similar to that in the control group. In group 3, slight but statistically significant reduction in food consumption was recorded from day 4 to 7 of the pre-pairing period. Afterwards, food consumption recovered and was similar to the control during the remaining study period. In group 2, food consumption was similar to thatin the control group during the entire study.

Females: Pre-Pairing, Gestation and Lactation Periods
Treatment with the test item caused a dose-dependent, reversible reduction in food consumption in groups 3 and 4. The effects in females were less pronounced than in males. In order of ascending dose levels, mean food consumption was 15.2, 16.0, 14.8 and 12.0 g/animal/day during pre-pairing period, 21.2, 21.3, 21.2 and 20.1 g/animal/day during the gestation period and 28.8, 26.2, 30.1 and 28.6 g/animal/day during the lactation period.
In group 4, reduction in food consumption was statistically significant from day 1 to 7 of the prepairing period and slight, not statistically significant until the end of thisperiod. During gestation and lactation periods food consumption was similar to that in the control group. In group 3, a slight but not statistically significant reduction was observed from day 1 to 7 of prepairing period and a recovery with slightly higher food consumption during the remaining prepairing period. Afterwards, similar or slightly higher values if compared to the controls were noted until the completion of the study. In group 2, food consumption was similar to thatin the control group during the entire study.
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
Males: Pre- and Post-Pairing Periods
Treatment with the test item caused a dose-dependent, reversiblereduction in food conversion efficiency in groups 3 and 4. In order of ascending dose levels, mean food conversion efficiency was 0.050, 0.050, 0.042 and 0.026 g bw/g food/animal/day during the pre-pairing period and 0.016, 0.022, 0.018 and 0.017 g bw/g food/animal/dayduring the post-pairing period. In group 4, the reduction in food conversion efficiency was statistically significant from day 4 to 10 of the pre-pairing period. Afterwards, food conversion efficiency recovered; it remained slightly, not statistically significantly lower until the end of the pre-pairing period and was similar or slightly higher than the control values during the post-pairing period. In group 3, a statistically significant reduction was recorded from day 4 to 7 of the pre-pairing period whereas during the post-pairing food conversion efficiency was similar to control values. In group 2, food conversion efficiency was similar or slightly higher than in the control group during the entire study.

Females: Pre-Pairing, Gestation and Lactation Periods
Treatment with the test item caused a reduction in food conversion efficiency in groups 3 and 4 at the beginning of the treatment and during gestation. In order of ascending dose levels, mean food conversion efficiency was 0.034, 0.035, 0.029 and 0.027 g bw/g food/animal/day during the pre-pairing period, 0.288, 0.269, 0.260 and 0.230 g bw/g food/animal/day during the gestation period and 0.190, 0.280, 0.256 and 0.111 g bw/g food/animal/day during the lactation period.
In group 4, reduction in food conversion efficiency was statistically significant from day 1 to 4 of the pre-pairing period. During the remaining pre-pairing period differences observed in this group were not statistically significant. A reduction in food conversion efficiency was recorded again during the gestation period with a statisticalsignificance from day 7 to 20 of this period. Also during the lactation period food conversion efficiency was lower than in the control group. In the presence of a high variability of individual values, the difference was not statistically significant. In group 3, statistically significant reduction in food conversion efficiency was recorded from day 1 to 4 of the pre-pairing period and no statistically significant differences were noted until the completion of this period. The food conversion efficiency was reduced again with a statistical significance from day 14 to 20 of the gestation period but recovered and was higher than the
control value during the lactation period. No statistically significant differences in relative food consumption were observed in group 2.
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
Males - Water consumption was not affectedby the treatment with the test item at any dose level. Mean water consumption in order of ascending dose levels was 31.0, 29.1, 31.3 and 31.6 g/animal/day during the pre-pairing period and 29.3, 27.2, 29.1 and 30.9 g/animal/day during the post-pairing period.

Females - Treatment with the test item caused an increase inwater consumption during the gestation period followed by a reduction during the lactation in group 4. Mean water consumption in order of ascending dose levels was 22.7, 24.2, 28.2 and 26.8 g/animal/day during pre-pairing period, 32.8, 36.0, 38.5 and 42.3 g/animal/day during gestation period, and 33.9, 32.6, 34.3 and 26.1 g/animal/day during lactation period. In group 4, increased water consumption was recorded during the gestation period compared to the control group, with statistical significance being attained on several days. Afterwards, water consumption decreased and during the lactation period it was statistically significantly lower than in the control group. During the pre-pairing period water consumption was higher than the control value but the differences were very variable, statistically significant only on days 13 - 14 and they were not clearly dose dependent. In group 3, increased water consumption was recorded during the gestation period, but attained statistical significance compared to the control only on days 14-15 ofgestation. During the lactation period water consumption was similar to that of the control group. Slightly higher water consumption was observed in this group during the pre-pairing period. However, the differences were very variable, statistically significant only on days 4 - 5 and not clearly dose dependent. In group 2 water consumption was similar to the control values during the entire study period.
Haematological findings:
no effects observed
Description (incidence and severity):
No test item-related effects on hematology parameters were noted in males or females at any dose level.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Males - Treatment with the test item caused an increase in activity of alkaline phosphatase in group 4. Mean activity of alkaline phosphatase was 105.2 U/L in group 4 versus 75.8 U/L in the control group. The difference was not statistically significant but the value at the high-dose level was slightly above of the historical control range and similar effect was observed in females. Therefore this effect was considered to be related to the treatment with the test item. The following statistically significant changes noted in males in group 4 were considered not to be related to the treatment: higher concentration of bilirubin (2.45 µmol/L compared to 0.0 µmol/L in the control group) and higher concentration of cholesterol (2.96 mmol/L compared to 1.98 mmol/L in the control group). Further, lower concentration of bile acids was noted in groups 3 and 4 (8.18 µmol/L and 7.72 µmol/L, respectively, compared to 19.74 µmol/L in the control group). These changes wereonly minor and values remained within or close to the range of historical controls, therefore they were considered to be a result of biological variability and not test item-related. No further statistically significant differences were observed in any group.

Females - Treatment with the test item caused a significant increase in activity of alkaline phosphatase and a higher concentration of triglycerides in group 4. Mean activityof alkaline phosphatase was 102.8 U/L in group 4 versus 52.3 U/L in the control group whereas mean concentration of triglycerides was 3.44 versus 0.93 mmol/L in the control group. Both values at the high-dose level were above of the historical control range (from 20.8 to 75.7 for 95% tolerance range for
alkaline phosphatase and 0.75 ± 0.48 mmol/L for triglycerides). Further statistically significant changes noted in females were considered not to be related to the treatment: higher concentration of urea (10.96 compared to 6.99 mmol/L), higher concentration of creatinine (36.3 compared to27.7 µmol/L), higher concentration of cholesterol (3.00 compared to 2.13 mol/L) and higher concentration of potassium (4.75 compared to 4.15 mmol/L for potassium). These changes were only minor and values remained within or close to the range of historical controls, therefore they were considered to be a result of biological variability. No further statistically significant differences were observed in any group.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
No test item-related findings were recorded during functional observational battery in males or females in any group.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Treatment with the test item caused an increase in liver weights in males and females in groups 3 and 4. In group 3, an increase in liver weight relative to body weight was statistically significant compared to the control group in both sexes. Absolute liver weights and liver weight to brain weight ratios were higher than control values but the differences were not statistically significant.Remaining statistically significant differences were considered not to be related to the treatment with the test item. In particular, changes in heart, adrenal gland, testes and epididymides weights were considered to be secondary to a reduction in body weights in both males and females in the high-dose group. In group 2, statistically significant increase in thymus weights and thymus weights to brain weight ratio was recorded in absence of any effect on thymus weight in animals at the higher dose levels.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No findings, which were considered to be test item related, were noted in any group.
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
The repeated oral administration ofthe test item in Han Wistar rats over a period of a minimum of five weeks at the doses of 2500, 5000 and 10000 ppm did not induce any test item-related adverse pathology findings in males or in females, including within reproductive organs. The only test-item finding was minimal hypertrophy/vacuolation of the zona glomerulosa in the adrenals glands at the high dose of 10000 ppm.
Other effects:
no effects observed
Description (incidence and severity):
Locomotor activity was similar in all groups in males and females.
Reproductive function: oestrous cycle:
effects observed, treatment-related
Description (incidence and severity):
Corpora Lutea Count:
The mean number of corpora lutea per dam was 15.5, 14.9, 14.2 and 12.5 in groups 1, 2, 3 and 4, respectively. The mean number of corpora luteaat the high-dose level was statistically significantly lower if compared to the control value. This was consideredto be related to the treatment with the test item.

Duration of Gestation:
Duration of gestation was unaffected by exposure tothe test item.

Implantation Rate and Post-Implantation Loss:
The mean number of implantationsper dam and post-implantation loss were considered not to be affected by the treatment with the test item.
Reproductive performance:
no effects observed
Description (incidence and severity):
With exception for two females in group 2, all females mated within the first pairing period. Percentage mating (number of animals mated as a percentage of animals paired) was 83% for males in group 2 and 100% in the remaining groups and for females 100% in all groups. Fertility index (number of females achieving pregnancy as a percentage of females paired), conception rate (number of females achieving pregnancy as a percentage of females mated) and gestation index (number of females with living pups as a percentage of females pregnant) were 100% in all groups.
Litter Size at First Litter Check:
The mean number of pups per dam at first litter check was 11.9, 11.6, 10.9 and 10.1 in groups 1, 2, 3 and 4, respectively. The number of pups per dam decreased slightly with increasing dose level, the differences were however not statistically significant and in the range of historicalcontrols, therefore they were considered not to be related to the treatment with the test item.

Postnatal Loss Days 0 - 4 Post Partum:
Mean postnatal loss was 0.1, 0.6, 0.2 and 0.8 pups lost per dam whereas the total number of pups lost were 1, 7 (in 3 litters), 2 (in 1 litter) and 9 (in 3 litters), in group 1, 2, 3 and 4, respectively. Viability index (number of pups alive on day 4 post partum as a percentage of pups born alive) was 99.3%, 95.0%, 98.5% and 91.9% in order of ascending dose levels. in groups 2 and 4, the increase in total number of pups lost during lactation and the reduction in viability index were statistically significant whereas the differences in mean number of pups lost per litter as well as in the numberof affected litters were not statistically significant. This was due to an increased pup mortality observed in onlyone litter in each group. In group 4, five pups were missing on day 2 and two further pups were missing on day 4 post partumin litter no. 93. Increased mortality of pups was noted in one litter, no. 72, in group 2; one pup in this litter was missing on day 3 and four further pups were missing on day 4 post partum. Pups in these two litters had lower body weights if compared to the respective average value in the group. Pups in litter no. 93 did not accomplish the righting reflex test, which indicated their poor condition whereas some of pups in litter no. 72 were found without milk in the stomach and were cold to touch indicating neglect of the litter by the dam. Due to the incidental occurrence of increased post-implantation loss in only two individual litters as well as the lack of a dose dependency, this effect was considered not to be related to the treatment with the test item.
Dose descriptor:
NOEL
Effect level:
2 500 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects observed in male and female rats
Key result
Dose descriptor:
NOAEL
Effect level:
5 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
other: Based on body weight effects observed at the 10000 ppm concentration level.
Remarks on result:
other: Systemic Toxicity
Dose descriptor:
NOAEL
Effect level:
10 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Reproductive Toxicity
Clinical signs:
no effects observed
Description (incidence and severity):
No test item-related abnormal findings were noted in pups at first litter check or during lactation in any group.

One group 2 pup did not have milk in the stomach and was coldto the touch at the first litter check. In another litter in this group (no. 72, in which 5 pups were missing on day 4 post partum), pups that were cold to the touch and had no milk in their stomach were observed on days 3 and 4 post partum. No milk in the stomach was also observed for one pup in group 4 at the first litter check. For another pup in group 4 a missing tail apexwas recorded. None of these findings showed dose dependency and therefore theywere considered not to be related to the treatment with the test item.
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
Mean postnatal loss was 0.1, 0.6, 0.2 and 0.8 pups lost per dam whereas the total number of pups lost were 1, 7 (in 3 litters), 2 (in 1 litter) and 9 (in 3 litters), in group 1, 2, 3 and 4, respectively. Viability index (number of pups alive on day 4 post partum as a percentage of pups born alive) was 99.3%, 95.0%, 98.5% and 91.9% in order of ascending dose levels. In groups 2 and 4, the increase in total number of pups lost during lactation and the reduction in viability index were statistically significant whereas the differences in mean number of pups lost per litter as well as in the numberof affected litters were not statistically significant. This was due to an increased pup mortality observed in onlyone litter in each group. In group 4, five pups were missing on day 2 and two further pups were missing on day 4 post partumin litter no. 93. Increased mortality of pups was noted in one litter, no. 72, in group 2; one pup in this litter was missing on day 3 and four further pups were missing on day 4 post partum. Pups in these two litters had lower body weights if compared to the respective average value in the group. Pups in litter no. 93 did not accomplish the righting reflex test, which indicated their poor condition whereas some of pups in litter no. 72 were found without milk in the stomach and were cold to touch indicating neglect of the litter by the dam. Due to the incidental occurrence of increased post-implantation loss in only two individual litters as well as the lack of a dose dependency, this effect was considered not to be related to the treatment with the test item.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Treatment with the test item resulted in reduced pup body weights and reduced pup body weight gain in group 4. Mean body weights of pups on day 1 post partum was 5.98, 5.62, 6.05 and 5.39 g, whereas body weight gain by day 4 post partum was 42.0%, 40.2%, 44.5% and 32.9%, in order of ascending dose levels. In group 4, lower body weights were recorded on days 1 and 4 post partumwith statistical significance for male pup body weights on day 4 post partum. Also body weight gain of pups was lower in this group but this was not statistically significant.

Although the reduction in pup body weights at the high-dose level was mostly not statistically significant if compared to the control group, values in group 4 were also reduced if compared to the historical controls and therefore this observation was considered to be related to the treatment.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No macroscopical findings were notedduring necropsy of pups in any group.
Other effects:
no effects observed
Description (incidence and severity):
Sex ratios at first litter check and on day 4 post partumwere unaffected by exposure to the test item.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Righting Reflex:
Percentages of pups for which righting reflex was observed within the first one minute of the test were 93.6%, 94.6%, 96.1% and 87.7% in groups 1, 2, 3 and 4, respectively. In group 4, the percentage of pups, for which righting reflex was observed, was reduced if compared to the control group, 14% (16 pups of 111) compared to 7% (10 of 143 pups) did not accomplish the test. Ten of the pups in group 4 with the negative result of the test were from one litter (no. 93). Due to the incidental occurrence in only one litter, worse performance of pupswas considered not to be related to the treatment with the test item.
Dose descriptor:
NOEL
Generation:
F1
Effect level:
5 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Reproductive/Developmental Toxicity
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
10 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Reproductive/Developmental Toxicity
Key result
Reproductive effects observed:
no

Table 4. BODY WEIGHTS (GRAM) OF MALES SUMMARY P GENERATION - PRE-PAIRING PERIOD

 

 

Group 1

0 ppm

Group 2

2500 ppm

Group 3

5000 ppm

Group 4

10000 ppm

 

Day 1

Mean

331

329

334

333

 

ST. Dev

10.7

10.2

11.8

8.6

 

N

12

12

12

12

 

Day 4

Mean

333

332

339

321*

 

ST. Dev

11.0

12.2

11.7

8.3

 

N

12

12

12

12

 

Day 7

Mean

351

349

349

329**

 

ST. Dev

11.4

14.7

12.6

8.1

 

N

12

12

12

12

 

Day 10

Mean

363

361

358

332**

 

ST. Dev

11.9

15.9

13.0

10.8

 

N

12

12

12

12

 

Day 13

Mean

372

371

367

339**

 

ST. Dev

12.7

17.0

12.6

10.2

 

N

12

12

12

12

* / **: Dunnett-Test based on pooled variance significant at 5% (*) or 1% (**) level

Table 5. BODY WEIGHTS (GRAM) OF MALES SUMMARY P GENERATION - PAIRING

 

 

Group 1

0 ppm

Group 2

2500 ppm

Group 3

5000 ppm

Group 4

10000 ppm

 

Day 4

Mean

376

380

369

345**

 

ST. Dev

13.7

16.9

16.7

10.6

 

N

12

12

12

12

 

Day 8

Mean

391

393

384

359**

 

ST. Dev

15.2

17.5

14.6

12.5

 

N

12

12

12

12

 

Day 15

Mean

408

408

399

375**

 

ST. Dev

17.1

18.8

18.6

14.0

 

N

12

12

12

12

* / **: T-Test based on pooled variance significant at 5% (*) or 1% (**) level

Table 6. BODY WEIGHTS (GRAM) OF MALES SUMMARY P GENERATION - POST-PAIRING

 

 

Group 1

0 ppm

Group 2

2500 ppm

Group 3

5000 ppm

Group 4

10000 ppm

 

Day 1

Mean

411

411

401

377**

 

ST. Dev

19.1

18.8

19.0

14.4

 

N

12

12

12

12

 

Day 4

Mean

416

416

406

382**

 

ST. Dev

17.4

18.1

19.3

15.4

 

N

12

12

12

12

 

Day 7

Mean

422

425

412

387**

 

ST. Dev

18.2

20.3

17.2

16.7

 

N

12

12

12

12

 

Day 10

Mean

425

429

414

389**

 

ST. Dev

15.8

21.7

18.5

14.9

 

N

12

12

12

12

 

Day 12

Mean

423

427

415

390**

 

ST. Dev

15.5

23.1

18.2

14.9

 

N

12

12

12

12

* / **: Dunnett-Test based on pooled variance significant at 5% (*) or 1% (**) level

Table 7. BODY WEIGHTS (GRAM) OF FEMALES SUMMARY P GENERATION - PRE-PAIRING PERIOD

 

 

Group 1

0 ppm

Group 2

2500 ppm

Group 3

5000 ppm

Group 4

10000 ppm

 

Day 1

Mean

196

198

200

199

 

ST. Dev

11.3

9.1

9.6

9.7

 

N

12

12

12

12

 

Day 4

Mean

199

204

201

187*

 

ST. Dev

11.8

9.9

11.7

7.1

 

N

12

12

12

12

 

Day 7

Mean

204

208

205

192*

 

ST. Dev

12.1

10.5

12.1

9.5

 

N

12

12

12

12

 

Day 10

Mean

208

213

209

195*

 

ST. Dev

12.4

11.3

10.6

14.7

 

N

12

12

12

12

 

Day 13

Mean

214

219

216

203

 

ST. Dev

12.2

11.4

12.3

10.8

 

N

12

12

12

12

* / **: Dunnett-Test based on pooled variance significant at 5% (*) or 1% (**) level

Table 8. BODY WEIGHTS (GRAM) OF FEMALES SUMMARY P GENERATION - GESTATION

 

 

Group 1

0 ppm

Group 2

2500 ppm

Group 3

5000 ppm

Group 4

10000 ppm

 

Day 0

Mean

218

233*

218

208

 

ST. Dev

14.0

16.6

14.2

11.9

 

N

12

12

12

11

 

Day 7

Mean

243

255

244

233

 

ST. Dev

15.7

15.0

15.6

10.7

 

N

12

12

12

11

 

Day 14

Mean

273

283

272

252*

 

ST. Dev

20.1

15.9

16.8

12.2

 

N

12

12

12

11

 

Day 20

Mean

338

345

327

299**

 

ST. Dev

26.5

20.8

24.7

18.3

 

N

12

12

12

11

* / **: Dunnett-Test based on pooled variance significant at 5% (*) or 1% (**) level

Table 9. BODY WEIGHTS (GRAM) OF FEMALES SUMMARY P GENERATION - LACTATION

 

 

Group 1

0 ppm

Group 2

2500 ppm

Group 3

5000 ppm

Group 4

10000 ppm

 

Day 1

Mean

254

247

240

226**

 

ST. Dev

24.6

19.4

16.1

16.1

 

N

12

12

12

11

 

Day 4

Mean

270

266

263

233**

 

ST. Dev

20.5

23.2

14.1

14.0

 

N

12

12

12

11

* / ** : Dunnett-Test based on pooled variance significant at 5% (*) or 1% (**) level

Table 10. REPRODUCTION and BREEDING DATA – P GENERATION

 

 

Group 1

0 ppm

Group 2

2500 ppm

Group 3

5000 ppm

Group 4

10000 ppm

 

Litters

Total

12

12

12

11

 

Excluded

-

-

-

-

 

Evaluated

12

12

12

11

 

Duration of Gestation

Mean (+)

21.4

21.6 -

21.7 -

21.2 -

 

ST. Dev

0.5

0.5

0.5

0.4

 

N

12

12

12

11

 

Corpora Lutea

Total

186

179

170

137

 

Mean (+)

15.5

14.9 -

14.2 -

12.5 +

 

ST. Dev

2.6

1.6

2.8

1.9

 

N

12

12

12

11

 

Implantations

Total

151

164

145

122

 

Mean (+)

12.6

13.7 -

12.1 -

11.1 -

 

ST. Dev

1.8

1.8

2.2

2.2

 

N

12

12

12

11

 

Post Implantation Loss

% of Implantations

5.3

15.2

9.7

9.0

 

Litter affected (#)

4

6 -

9 -

7 -

 

Total (#)

8

25 ##

14 -

11 -

 

Mean (+)

0.7

2.1 -

1.2 -

1.0 -

 

ST. Dev

1.4

4.2

0.9

1.0

 

N

12

12

12

11

 

Dead Pups at First Litter Check

Litters affected (#)

-

1 -

-      -

2 -

 

Total (#)

0

14

0

3

 

Mean (+)

0.0

1.2 -

0.0 -

0.3 -

 

ST. Dev

0.0

4.0

0.0

0.6

 

N

12

12

12

11

#/##/- : Fisher's Exact Test, significant at 5% (#), 1% (##) or not significant (-)

+/++/-: Steel Test, significant at 5% (+), 1% (++) or not significant (-)

Conclusions:
The NOEL (No Observed Effect Level) was established at the concentration level of 2500 ppm and the NOAEL (No Observed Adverse Effect Level) was established at 5000 ppm for general toxicity. For reproductive and developmental toxicity, the NOEL was established at 5000 ppm whereas the NOAEL was established at the concentration level of 10000 ppm, the highest concentration tested in the study.
Executive summary:

In a key combined repeated dose, reproductive/developmental toxicity screening study, the test material (Tall oil rosin, CAS# 8050-09-7) was administered daily in dietary mixtures at concentrations of 0, 2500 ppm (males - 160 mg/Kg bw/day Pre-Pairing and 136.4 mg/Kg bw/day Post-Pairing; females - 189 mg/Kg bw/day Pre-Pairing; 182.8 mg/Kg bw/day Gestation; and 241.1 mg/K bw/day Lactation), 5000 ppm (males - 301.5 mg/Kg bw/day Pre-Pairing and 276.5mg/K bw/day Post-Pairing; females – 356.1 mg/Kg bw/day Pre-Pairing; 377.3 mg/Kg bw/day Gestation; and 572.7 mg/K bw/day Lactation), or 10000 ppm (males - 496.4 mg/Kg bw/day Pre-Pairing and 585.5 mg/K bw/day Post-Pairing; females – 612.9 mg/Kg bw/day Pre-Pairing; 772.2 mg/Kg bw/day Gestation; and 1227.9 mg/K bw/day Lactation) to male rats for 42 days and to female rats for 14 days prior to pairing, through the pairing and gestation periods until the F1 generation reached day 4 post partum.

 

Treatment resulted in slightly higher test item intake in females compared to males during the pre-pairing period. Intake remained fairly constant throughout the entire study, except during the lactation phase when, due to an increase in food consumption, intake in females was almost two-fold higher than during the preceding gestation period. In the high concentration group (10000 ppm), a reduction in food consumption resulting in body weight loss followed by reduced body weight gain, significantly reduced body weights, and significantly reduced food conversion efficiency were observed in male and female rats. A recovery was observed for food consumption in males and females and for body weight gain and food conversion efficiency in males. In females, body weight gain and food conversion efficiency recovered also initially but both values reduced again during the gestation period and remained lower than the control values during the lactation period. Body weights in both males and females, remained reduced compared to the controls during the entire study. Body weight loss in one female led to premature termination of the animal for humane reasons. In females, effects on water consumption were also noted in the 10000 ppm group; an increase was noted during the gestation period followed by a reduction during the lactation period. Due to the recovery observed during the study and because Tall Oil Rosin (CAS # 8050-09-7) is known to cause palatability problems, effects on food consumption were considered to most probably not be a sign of toxic potential but resulted from unpalatability of the test item. The lower body weights in males of the 10000 ppm group compared to concurrent control throughout the study was considered to represent impaired growth resulting from food aversion during the early

treatment period. Similarly, lower body weights in females were considered to result from undernutrition at the beginning of the study. In females, however, a second drop in body weight gain and food consumption efficiency was observed during the gestation period after the recovery of food consumption. These later effects were considered to be related to potential toxicity of the test item. Reduction in body weights in both genders and reductions in body

weight gain and food conversion efficiency in females at the high concentration level were considered to be adverse because they were not reversible during the course of the study.

 

Further, terminal examinations revealed increased liver weights in males and females in the 10000 ppm group. This effect was accompanied by an increase in alkaline phosphatase activity but no changes in the blood activity of other liver enzymes were observed and there were no histopathological lesions in this organ in any gender. For these reasons, increased liver weights were considered not to be adverse and probably a result of increased metabolism due to the exposure to foreign compound.

 

In the high concentration group, the number of corpora lutea was reduced, which resulted in slightly, not statistically significant lower number of implantation sites and slightly, not statistically significant lower litter size at first litter check. Mean litter size at the high concentration level remained in the range of historical control values. Slightly reduced body weight and body weight gain were observed in pups during lactation. Although the reduction was only minor and predominantly not statistically significant if compared to the current control, values at the high concentration level were lower if compared to the historical control background and therefore this observation was considered to

be related to the treatment with the test item.

 

Mating performance, fertility, duration of gestation, post-implantation and post-natal loss were considered not to be affected by the treatment with the test item at any concentration level. Due to adverse effects on body weight observed in females in the high concentration group, a possibility that the reduced number of corpora lutea as well as lower pup body weight were secondary to the maternal response, should be taken into consideration.

 

In the intermediate concentration group (5000 ppm), a slight reduction in food consumption was observed in both genders at the beginning of the treatment. In males, this resulted in slight reductions in body weight gain and food conversion efficiency at the beginning of the treatment and slightly, not statistically significant, lower body weights during the entire study. In females, body weight gain and food conversion efficiency were also slightly reduced at the beginning of the study and recovered thereafter whereas body weights were not changed compared to the control values during the entire study. Food conversion efficiency reduced again during the gestation period but recovered and was higher than the control value during the lactation period. Water consumption was slightly and reversibly increased in females of this group. Due to reversibility, effects on food consumption, body weights and water consumption in the intermediate concentration group were considered not to be adverse. Although food conversion efficiency was significantly lower in this group again during the gestation period, it recovered during the lactation period and did not correlate with reduced body weight gain or reduced body weights and therefore this effect was also considered not to be adverse.

 

In the 5000 ppm group, increased liver weights were observed in males and females but without histopathological lesions or changes in related clinical biochemistry parameters and therefore considered not to be adverse. No effects on reproductive or developmental parameters were observed in animals in the 5000 ppm group.

 

In the low concentration group (2500 ppm), no signs of general toxicity in males or females as well as no effects on reproduction or development were observed.

 

Based on these observations, the NOEL (No Observed Effect Level) was established at the concentration level of 2500 ppm and the NOAEL (No Observed Adverse Effect Level) was established at 5000 ppm for general toxicity. For reproductive and developmental toxicity, the NOEL was established at 5000 ppm whereas the NOAEL was established at the concentration level of 10000 ppm, the highest concentration tested in the study.

Reason / purpose:
reference to same study
Remarks:
Sections 7.5.1. and 7.8.2

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2016

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
The deviations did not impact the integrity and validity of the study
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
other: Pale yellow glassy solid

Test animals

Species:
rat
Strain:
other: RccHan TM : WIST(SPF)
Details on species / strain selection:
Recognized by international guidelines as a recommended test system.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories, B.V. Kreuzelweg 53, 5961 NM Horst / Netherlands
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: (P) 12 wks
- Weight at study initiation: (P) Males: 314 - 363 g; Females: 178 - 223 g
- Fasting period before study: Not specified
- Housing: During acclimatization and pre-pairing in groups of up to three animals divided by sex in Makrolon type-4 cages. Cages with males were interspersed amongst those holding females to promote the development of regular estrus cycles during pre-pairing period. During pairing females were housed with males (1:1) in Makrolon type-3 Following the pairing period males were re-housed in their original pre-pairing cages and females were individually housed in Makrolon type-3 cages. All cages were provided with wire mesh tops and sterilized standard softwood bedding (‘Lignocel’ J. Rettenmaier & Söhne GmbH & CoKG, 73494 Rosenberg / Germany, imported by Provimi Kliba SA, 4303 Kaiseraugst / Switzerland) with paper enrichment (Enviro-dri from Lillico, Biotechnology, Surrey / UK and ISO-BLOX from Harlan Laboratories B.V. / Netherlands). In addition a wood stick was provided (batch nos. 02105130819, 77, 6960C-100267 and 132211).
- Diet (e.g. ad libitum): Microgranulated standard Harlan Teklad 2018C (batch no. 41/13) rodent maintenance diet (Provimi Kliba SA, 4303 Kaiseraugst / Switzerland) was available ad libitum.
- Water (e.g. ad libitum): Community tap-water from Itingen was available ad libitum in water bottles.
- Acclimation period:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 30 - 70%
- Air changes (per hr): 10 - 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12-hour fluorescent light / 12-hour dark cycle with at least eight hours music during the light period.

IN-LIFE DATES: From: 2013-10-22 To: 2013-12-23

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
DIET PREPARATION

- Mixing appropriate amounts with (Type of food): Dietary admixtures were prepared at intervals not exceeding 28 days using the test item as supplied by the supplier. A portion of Tall Oil Rosin, CAS no. 8050-09-7, was ground to powder using an electrical
grinder, weighed into a tared glass beaker on a suitable precision balance, and mixed with microgranulated feed separately for each dose group. No heat was used during diet preparation. Control feed for the animals of group 1 was prepared similarly, but without test item.
- Storage temperature of food: Stability of test item in feed was at least 28 days upon the results of stability analyses performed within the Harlan Laboratories study no. D80904 (Method Implementation and Development). Feed preparations were stored at room temperature protected from light in metal containers until use.
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: until evidence of copulation was observed
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: Not specified
- After successful mating each pregnant female was caged (how): Individually
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples for analyses of test item, its content and homogeneity in the feed were drawn at the start of the pre-pairing period, at second occasion of dietary admixture preparation and at the end of gestation/start of lactation period (i.e. three occasions). Stability of the test item in the feed for 28 days at room temperature was determined at the second occasion of dietary admixture preparation (i.e. one occasion). For assessment of content and homogeneity, samples of approximately 100 g were collected from each the top, middle and bottom of the container of every dietary admixture of the respective diet preparation (from group 1 admixtures only one sample was drawn from the middle of the preparation). Samples for assessment of content and homogeneity were collected on the day of preparation. For assessment of stability, samples of approximately 100 g were drawn from the middle of every dietary admixture on the day of preparation and stored at room temperature for 28 days. After preparation the samples were delivered to the analytical department at ambient temperature and analyzed immediately or stored there frozen (at -20 ± 5 °C) until analysis. The samples were analyzed by HPLC coupled toan ELSD detector following an analytical procedure developed at Harlan Laboratories. The test item was usedas the analytical standard. Duplicates were taken of all samples and were stored at Harlan Laboratories Ltd., Itingen / Switzerland. The samples were discarded after finalization of the report without any further note.
Duration of treatment / exposure:
Males: 42 days (during 14 days pre-pairing period, up to 28 days pairing period and post-pairing period).
Females: Minimum 5 weeks (14 days pre-pairing period, up to 28 days pairing period, approximately 21 days of gestation period and lactation period to day 4 post partum).
Frequency of treatment:
Continuously (ad libitum)
Doses / concentrationsopen allclose all
Dose / conc.:
0 ppm
Remarks:
Control
Dose / conc.:
2 500 ppm
Remarks:
Low Concentration
Dose / conc.:
5 000 ppm
Remarks:
Intermediate Concentration
Dose / conc.:
10 000 ppm
Remarks:
High Concentration
No. of animals per sex per dose:
12/sex/dose
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: The dose levels were selectedbased on a previous dose range-finding toxicity study in Han Wistar rats, using test material from the same batch, Harlan Laboratories study no. D80915, using concentrations in feed of 5000, 10000 and 20000 ppm and resulting in adverse reduction of food consumption, body weight loss and early termination of males and females treated with feed containing test item at concentration of 20000 ppm. A dietary inclusion level of 10000 ppm resulted in approximately 10% reduction in body weight compared to control during 15 days of treatment.
- Rationale for animal assignment (if not random): Performed after at least three days of acclimatization using a computer-generatedrandom algorithm. Body weights (recorded on the day of allocation) were taken into consideration in order to ensure similar
mean body weights in all groups.

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily cage-side clinical observations (once daily, during acclimatization and up to day of necropsy).

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Males: Once prior to the first administration of the test item and weekly thereafter.
Females: Once prior to the first administration of the test item, weekly during the pre-pairing and pairing and on days 0, 6, 13 and 20 post coitum.

BODY WEIGHT: Yes
- Time schedule for examinations: Males: Once during acclimatization, at three-day intervals during pre-pairing and post-pairing periods and weekly during pairing period.
Females: Once during acclimatization, at three-day intervals during pre-pairing period, weekly during pairing period and on the following days: 0, 7, 14 and 20 post coitumand 1 and 4 post partum.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
Males: At three-day intervals during pre-pairing and post-pairing.
Females: At three-day intervals during pre-pairing and for periods: days 0 - 7, 7 - 14 and 14 -20 post coitum and 1 - 4 post partum. No food consumption was recorded during the pairing period.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: Daily water intake was measured gravimetrically.

Litter observations:
PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, Surface righting reflex, Functional Observation Battery,

GROSS EXAMINATION OF DEAD PUPS: yes, for external and internal abnormalities

Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals - Males were sacrificed on the day 43, when theywere no longer necessary for the assessment of reproductive performance.
- Maternal animals: All surviving animals - Damswere sacrificed on day 5 post partum.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations - All animals surviving to the end of the observation period and all moribund animals were anesthetized by intraperitonealinjection of pentobarbitoneand killed by exsanguinations. All parent animals and pups, except those excessively cannibalized, were examined macroscopically for any structural changes,either at the scheduled necropsy or during the study if death occurred prior to scheduled sacrifice. For the parent animals, special attention was directed at the organs of the reproductive system. The uteri of all dams were placed in a solution of ammonium sulfide to visualize possible hemorrhagic areas of implantation sites and the number of implantation sites was noted. The number of corpora luteain each ovary was recorded for all pregnant females.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table 2 and 3 were prepared for microscopic examination and weighed, respectively.
Postmortem examinations (offspring):
SACRIFICE
- The F1 Pups were sacrificed on day 4 post partum.
Statistics:
The following statistical methods were used to analyze food consumption, body weights water consumption, clinical laboratory data, grip strength, locomotor activity, organ weights, macroscopic findings and reproduction data:

• Means and standard deviationsof various data were calculated and included in the report.
• The Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groupsand the control groups for each sex.
• The Steel-test (many-to-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.
• Fisher's exact-test was applied if the variables could be dichotomized without loss of information.
Reproductive indices:
1) Mating Performance and Fertility
2) Corpora Lutea Count
3) Duration of Gestation
4) Implantation Rate and Post-Implantation Loss
5) Litter Size at First Litter Check
6) Postnatal Loss Days 0 - 4 Post Partum
Offspring viability indices:
1) Litter Size at First Litter Check
2) Postnatal Loss Days 0 - 4 Post Partum
3) Sex ratios
4) Righting Reflex

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No test item related clinical signs wereobserved in males or females in any group. In group 2, one female (no. 64)had hunched posture and chromodacryorrhea on days 1 and 2 as well as ruffled fur from day 1 to 3 post partum. Due to isolated occurrence in the low-dose group, these observations were considered not to be related to the treatment with the test item.
Mortality:
mortality observed, treatment-related
Description (incidence):
With exception for one female in group 4, all animals survived until the scheduled necropsy. In group 4, body weight loss up to 24% was noted in one female (no. 85) from the beginning of treatment. This female was terminated for ethical reasons on day 12 of the pre-pairing period. No findings were noted during necropsy of this female. The early termination was considered to be a result of the treatment with the test item.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Males - Pre-Pairing, Pairing and Post-Pairing Periods
Treatment with the test item caused dose-dependent reduction in body weight and body weight gain in groups 3 and 4. Mean body weight was greater than 90% of the control group value throughout the treatment period. Mean body weight gain in order of ascending dose levels was 12.4%, 12.6%, 9.7% and 1.8% during the pre-pairing period, 8.2%, 7.5%, 8.1% and 8.4% during the pairing period, and 2.9%, 4.0%, 3.4% and 3.4% during the post-pairing period. In group 4, body weight loss up to 3.5% was noted on day 4 of the pre-pairing period followed by statistically significantly reduced body weight gain recorded until the end of this period. Body weight gain during the pairing and the post-pairing periods was similar to the control values (if expressed as a percentage of body weights in the group at the beginning of the respective period). Consequently, lower body weights compared to control were recorded from day 4 of the prepairing period, and which remained approximately 91-92% of the mean control group body weight from day 10 until the completion of the study. This reduction was statistically significant during the entire study period. In group 3, statistically significantly reduced body weight gain was noted from day 7 to 13 of the pre-pairing period and body weight gain similar to that recorded in the control group during the remaining study period. This resulted in slightly, not statistically significantly, lower body weights recorded from day 7 ofthe pre-pairing period onwards. In group 2, body weights and body weight gain weresimilar to the respective values in the control group.

Females - Pre-Pairing, Gestation and Lactation Periods
Treatment with the test item caused a reduction in body weights and body weight gain in group 4. Mean body weight gain in order of ascending dose levelswas 9.4%, 10.2%, 8.0% and 2.8% during the pre-pairing period, 55.1%, 48.6%, 49.8%and 43.8% during the gestation period, and 6.5%, 7.9%, 9.7% and 3.6% during the lactation period. Mean values ofbody weights and body weight gain were not calculated for females during the pairing period due to different length of this period for individual females. In group 4, body weight loss up to 5.8% was recorded on day 4 of the pre-pairing period and reduced body weight gain until the completion of the study. This reduction was statistically significant during the entire pre-pairing period and on days 14 and 20 of the gestation period. Consequently, reduced body weights were recordedfrom day 4 of the pre-pairing period until the completion of the study with statistical significance on the most ofthe days. Nevertheless, mean
bodyweight was no less than 94% ofthe control value throughout the pre-pairing period and the first week of the gestation period, but fell to 86% on lactation day 4. In groups 2 and 3, body weight and body weight gain were considered not to be affected by the treatment. In group 2 body weights and body weight gain were slightly higher than the respective control values during the entire study, with statistical significance on day 0 post coitum. The differences observed in this group were considered to be due to biological variability. In group 3, slightly, not statistically significantly lower body weight gain was noted during the pre-pairing and gestation periods. Body weights remained similar to the control values.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Males: Pre- and Post-Pairing Periods
Treatment with the test item caused a dose-dependent, reversible reduction in food consumption in groups 3 and 4. In order of ascending dose levels, mean food consumption was 22.3, 22.6, 21.3 and 16.5 g/animal/day during the pre-pairing period and 22.8, 23.2, 22.8 and 22.7 g/animal/day during the post-pairing period. In group 4, reduction in food consumption was statistically significant during the entire prepairing period. During the post-pairing period, food consumption in this group recovered and was similar to that in the control group. In group 3, slight but statistically significant reduction in food consumption was recorded from day 4 to 7 of the pre-pairing period. Afterwards, food consumption recovered and was similar to the control during the remaining study period. In group 2, food consumption was similar to thatin the control group during the entire study.

Females: Pre-Pairing, Gestation and Lactation Periods
Treatment with the test item caused a dose-dependent, reversible reduction in food consumption in groups 3 and 4. The effects in females were less pronounced than in males. In order of ascending dose levels, mean food consumption was 15.2, 16.0, 14.8 and 12.0 g/animal/day during pre-pairing period, 21.2, 21.3, 21.2 and 20.1 g/animal/day during the gestation period and 28.8, 26.2, 30.1 and 28.6 g/animal/day during the lactation period.
In group 4, reduction in food consumption was statistically significant from day 1 to 7 of the prepairing period and slight, not statistically significant until the end of thisperiod. During gestation and lactation periods food consumption was similar to that in the control group. In group 3, a slight but not statistically significant reduction was observed from day 1 to 7 of prepairing period and a recovery with slightly higher food consumption during the remaining prepairing period. Afterwards, similar or slightly higher values if compared to the controls were noted until the completion of the study. In group 2, food consumption was similar to thatin the control group during the entire study.
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
Males: Pre- and Post-Pairing Periods
Treatment with the test item caused a dose-dependent, reversiblereduction in food conversion efficiency in groups 3 and 4. In order of ascending dose levels, mean food conversion efficiency was 0.050, 0.050, 0.042 and 0.026 g bw/g food/animal/day during the pre-pairing period and 0.016, 0.022, 0.018 and 0.017 g bw/g food/animal/dayduring the post-pairing period. In group 4, the reduction in food conversion efficiency was statistically significant from day 4 to 10 of the pre-pairing period. Afterwards, food conversion efficiency recovered; it remained slightly, not statistically significantly lower until the end of the pre-pairing period and was similar or slightly higher than the control values during the post-pairing period. In group 3, a statistically significant reduction was recorded from day 4 to 7 of the pre-pairing period whereas during the post-pairing food conversion efficiency was similar to control values. In group 2, food conversion efficiency was similar or slightly higher than in the control group during the entire study.

Females: Pre-Pairing, Gestation and Lactation Periods
Treatment with the test item caused a reduction in food conversion efficiency in groups 3 and 4 at the beginning of the treatment and during gestation. In order of ascending dose levels, mean food conversion efficiency was 0.034, 0.035, 0.029 and 0.027 g bw/g food/animal/day during the pre-pairing period, 0.288, 0.269, 0.260 and 0.230 g bw/g food/animal/day during the gestation period and 0.190, 0.280, 0.256 and 0.111 g bw/g food/animal/day during the lactation period.
In group 4, reduction in food conversion efficiency was statistically significant from day 1 to 4 of the pre-pairing period. During the remaining pre-pairing period differences observed in this group were not statistically significant. A reduction in food conversion efficiency was recorded again during the gestation period with a statisticalsignificance from day 7 to 20 of this period. Also during the lactation period food conversion efficiency was lower than in the control group. In the presence of a high variability of individual values, the difference was not statistically significant. In group 3, statistically significant reduction in food conversion efficiency was recorded from day 1 to 4 of the pre-pairing period and no statistically significant differences were noted until the completion of this period. The food conversion efficiency was reduced again with a statistical significance from day 14 to 20 of the gestation period but recovered and was higher than the
control value during the lactation period. No statistically significant differences in relative food consumption were observed in group 2.
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
Males - Water consumption was not affectedby the treatment with the test item at any dose level. Mean water consumption in order of ascending dose levels was 31.0, 29.1, 31.3 and 31.6 g/animal/day during the pre-pairing period and 29.3, 27.2, 29.1 and 30.9 g/animal/day during the post-pairing period.

Females - Treatment with the test item caused an increase inwater consumption during the gestation period followed by a reduction during the lactation in group 4. Mean water consumption in order of ascending dose levels was 22.7, 24.2, 28.2 and 26.8 g/animal/day during pre-pairing period, 32.8, 36.0, 38.5 and 42.3 g/animal/day during gestation period, and 33.9, 32.6, 34.3 and 26.1 g/animal/day during lactation period. In group 4, increased water consumption was recorded during the gestation period compared to the control group, with statistical significance being attained on several days. Afterwards, water consumption decreased and during the lactation period it was statistically significantly lower than in the control group. During the pre-pairing period water consumption was higher than the control value but the differences were very variable, statistically significant only on days 13 - 14 and they were not clearly dose dependent. In group 3, increased water consumption was recorded during the gestation period, but attained statistical significance compared to the control only on days 14-15 ofgestation. During the lactation period water consumption was similar to that of the control group. Slightly higher water consumption was observed in this group during the pre-pairing period. However, the differences were very variable, statistically significant only on days 4 - 5 and not clearly dose dependent. In group 2 water consumption was similar to the control values during the entire study period.
Haematological findings:
no effects observed
Description (incidence and severity):
No test item-related effects on hematology parameters were noted in males or females at any dose level.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Males - Treatment with the test item caused an increase in activity of alkaline phosphatase in group 4. Mean activity of alkaline phosphatase was 105.2 U/L in group 4 versus 75.8 U/L in the control group. The difference was not statistically significant but the value at the high-dose level was slightly above of the historical control range and similar effect was observed in females. Therefore this effect was considered to be related to the treatment with the test item. The following statistically significant changes noted in males in group 4 were considered not to be related to the treatment: higher concentration of bilirubin (2.45 µmol/L compared to 0.0 µmol/L in the control group) and higher concentration of cholesterol (2.96 mmol/L compared to 1.98 mmol/L in the control group). Further, lower concentration of bile acids was noted in groups 3 and 4 (8.18 µmol/L and 7.72 µmol/L, respectively, compared to 19.74 µmol/L in the control group). These changes wereonly minor and values remained within or close to the range of historical controls, therefore they were considered to be a result of biological variability and not test item-related. No further statistically significant differences were observed in any group.

Females - Treatment with the test item caused a significant increase in activity of alkaline phosphatase and a higher concentration of triglycerides in group 4. Mean activityof alkaline phosphatase was 102.8 U/L in group 4 versus 52.3 U/L in the control group whereas mean concentration of triglycerides was 3.44 versus 0.93 mmol/L in the control group. Both values at the high-dose level were above of the historical control range (from 20.8 to 75.7 for 95% tolerance range for
alkaline phosphatase and 0.75 ± 0.48 mmol/L for triglycerides). Further statistically significant changes noted in females were considered not to be related to the treatment: higher concentration of urea (10.96 compared to 6.99 mmol/L), higher concentration of creatinine (36.3 compared to27.7 µmol/L), higher concentration of cholesterol (3.00 compared to 2.13 mol/L) and higher concentration of potassium (4.75 compared to 4.15 mmol/L for potassium). These changes were only minor and values remained within or close to the range of historical controls, therefore they were considered to be a result of biological variability. No further statistically significant differences were observed in any group.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
No test item-related findings were recorded during functional observational battery in males or females in any group.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Treatment with the test item caused an increase in liver weights in males and females in groups 3 and 4. In group 3, an increase in liver weight relative to body weight was statistically significant compared to the control group in both sexes. Absolute liver weights and liver weight to brain weight ratios were higher than control values but the differences were not statistically significant.Remaining statistically significant differences were considered not to be related to the treatment with the test item. In particular, changes in heart, adrenal gland, testes and epididymides weights were considered to be secondary to a reduction in body weights in both males and females in the high-dose group. In group 2, statistically significant increase in thymus weights and thymus weights to brain weight ratio was recorded in absence of any effect on thymus weight in animals at the higher dose levels.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No findings, which were considered to be test item related, were noted in any group.
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
The repeated oral administration ofthe test item in Han Wistar rats over a period of a minimum of five weeks at the doses of 2500, 5000 and 10000 ppm did not induce any test item-related adverse pathology findings in males or in females, including within reproductive organs. The only test-item finding was minimal hypertrophy/vacuolation of the zona glomerulosa in the adrenals glands at the high dose of 10000 ppm.
Other effects:
no effects observed
Description (incidence and severity):
Locomotor activity was similar in all groups in males and females.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
effects observed, treatment-related
Description (incidence and severity):
Corpora Lutea Count:
The mean number of corpora lutea per dam was 15.5, 14.9, 14.2 and 12.5 in groups 1, 2, 3 and 4, respectively. The mean number of corpora luteaat the high-dose level was statistically significantly lower if compared to the control value. This was consideredto be related to the treatment with the test item.

Duration of Gestation:
Duration of gestation was unaffected by exposure tothe test item.

Implantation Rate and Post-Implantation Loss:
The mean number of implantationsper dam and post-implantation loss were considered not to be affected by the treatment with the test item.
Reproductive performance:
no effects observed
Description (incidence and severity):
With exception for two females in group 2, all females mated within the first pairing period. Percentage mating (number of animals mated as a percentage of animals paired) was 83% for males in group 2 and 100% in the remaining groups and for females 100% in all groups. Fertility index (number of females achieving pregnancy as a percentage of females paired), conception rate (number of females achieving pregnancy as a percentage of females mated) and gestation index (number of females with living pups as a percentage of females pregnant) were 100% in all groups.

Details on results (P0)

Litter Size at First Litter Check:
The mean number of pups per dam at first litter check was 11.9, 11.6, 10.9 and 10.1 in groups 1, 2, 3 and 4, respectively. The number of pups per dam decreased slightly with increasing dose level, the differences were however not statistically significant and in the range of historicalcontrols, therefore they were considered not to be related to the treatment with the test item.

Postnatal Loss Days 0 - 4 Post Partum:
Mean postnatal loss was 0.1, 0.6, 0.2 and 0.8 pups lost per dam whereas the total number of pups lost were 1, 7 (in 3 litters), 2 (in 1 litter) and 9 (in 3 litters), in group 1, 2, 3 and 4, respectively. Viability index (number of pups alive on day 4 post partum as a percentage of pups born alive) was 99.3%, 95.0%, 98.5% and 91.9% in order of ascending dose levels. in groups 2 and 4, the increase in total number of pups lost during lactation and the reduction in viability index were statistically significant whereas the differences in mean number of pups lost per litter as well as in the numberof affected litters were not statistically significant. This was due to an increased pup mortality observed in onlyone litter in each group. In group 4, five pups were missing on day 2 and two further pups were missing on day 4 post partumin litter no. 93. Increased mortality of pups was noted in one litter, no. 72, in group 2; one pup in this litter was missing on day 3 and four further pups were missing on day 4 post partum. Pups in these two litters had lower body weights if compared to the respective average value in the group. Pups in litter no. 93 did not accomplish the righting reflex test, which indicated their poor condition whereas some of pups in litter no. 72 were found without milk in the stomach and were cold to touch indicating neglect of the litter by the dam. Due to the incidental occurrence of increased post-implantation loss in only two individual litters as well as the lack of a dose dependency, this effect was considered not to be related to the treatment with the test item.

Effect levels (P0)

open allclose all
Dose descriptor:
NOEL
Effect level:
2 500 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects observed in male and female rats
Key result
Dose descriptor:
NOAEL
Effect level:
5 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
other: Based on body weight effects observed at the 10000 ppm concentration level.
Remarks on result:
other: Systemic Toxicity
Dose descriptor:
NOAEL
Effect level:
10 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Reproductive Toxicity

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Description (incidence and severity):
No test item-related abnormal findings were noted in pups at first litter check or during lactation in any group.

One group 2 pup did not have milk in the stomach and was coldto the touch at the first litter check. In another litter in this group (no. 72, in which 5 pups were missing on day 4 post partum), pups that were cold to the touch and had no milk in their stomach were observed on days 3 and 4 post partum. No milk in the stomach was also observed for one pup in group 4 at the first litter check. For another pup in group 4 a missing tail apexwas recorded. None of these findings showed dose dependency and therefore theywere considered not to be related to the treatment with the test item.
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
Mean postnatal loss was 0.1, 0.6, 0.2 and 0.8 pups lost per dam whereas the total number of pups lost were 1, 7 (in 3 litters), 2 (in 1 litter) and 9 (in 3 litters), in group 1, 2, 3 and 4, respectively. Viability index (number of pups alive on day 4 post partum as a percentage of pups born alive) was 99.3%, 95.0%, 98.5% and 91.9% in order of ascending dose levels. In groups 2 and 4, the increase in total number of pups lost during lactation and the reduction in viability index were statistically significant whereas the differences in mean number of pups lost per litter as well as in the numberof affected litters were not statistically significant. This was due to an increased pup mortality observed in onlyone litter in each group. In group 4, five pups were missing on day 2 and two further pups were missing on day 4 post partumin litter no. 93. Increased mortality of pups was noted in one litter, no. 72, in group 2; one pup in this litter was missing on day 3 and four further pups were missing on day 4 post partum. Pups in these two litters had lower body weights if compared to the respective average value in the group. Pups in litter no. 93 did not accomplish the righting reflex test, which indicated their poor condition whereas some of pups in litter no. 72 were found without milk in the stomach and were cold to touch indicating neglect of the litter by the dam. Due to the incidental occurrence of increased post-implantation loss in only two individual litters as well as the lack of a dose dependency, this effect was considered not to be related to the treatment with the test item.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Treatment with the test item resulted in reduced pup body weights and reduced pup body weight gain in group 4. Mean body weights of pups on day 1 post partum was 5.98, 5.62, 6.05 and 5.39 g, whereas body weight gain by day 4 post partum was 42.0%, 40.2%, 44.5% and 32.9%, in order of ascending dose levels. In group 4, lower body weights were recorded on days 1 and 4 post partumwith statistical significance for male pup body weights on day 4 post partum. Also body weight gain of pups was lower in this group but this was not statistically significant.

Although the reduction in pup body weights at the high-dose level was mostly not statistically significant if compared to the control group, values in group 4 were also reduced if compared to the historical controls and therefore this observation was considered to be related to the treatment.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No macroscopical findings were notedduring necropsy of pups in any group.
Other effects:
no effects observed
Description (incidence and severity):
Sex ratios at first litter check and on day 4 post partumwere unaffected by exposure to the test item.

Developmental neurotoxicity (F1)

Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Righting Reflex:
Percentages of pups for which righting reflex was observed within the first one minute of the test were 93.6%, 94.6%, 96.1% and 87.7% in groups 1, 2, 3 and 4, respectively. In group 4, the percentage of pups, for which righting reflex was observed, was reduced if compared to the control group, 14% (16 pups of 111) compared to 7% (10 of 143 pups) did not accomplish the test. Ten of the pups in group 4 with the negative result of the test were from one litter (no. 93). Due to the incidental occurrence in only one litter, worse performance of pupswas considered not to be related to the treatment with the test item.

Effect levels (F1)

open allclose all
Dose descriptor:
NOEL
Generation:
F1
Effect level:
5 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Reproductive/Developmental Toxicity
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
10 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Reproductive/Developmental Toxicity

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Any other information on results incl. tables

Table 4. BODY WEIGHTS (GRAM) OF MALES SUMMARY P GENERATION - PRE-PAIRING PERIOD

 

 

Group 1

0 ppm

Group 2

2500 ppm

Group 3

5000 ppm

Group 4

10000 ppm

 

Day 1

Mean

331

329

334

333

 

ST. Dev

10.7

10.2

11.8

8.6

 

N

12

12

12

12

 

Day 4

Mean

333

332

339

321*

 

ST. Dev

11.0

12.2

11.7

8.3

 

N

12

12

12

12

 

Day 7

Mean

351

349

349

329**

 

ST. Dev

11.4

14.7

12.6

8.1

 

N

12

12

12

12

 

Day 10

Mean

363

361

358

332**

 

ST. Dev

11.9

15.9

13.0

10.8

 

N

12

12

12

12

 

Day 13

Mean

372

371

367

339**

 

ST. Dev

12.7

17.0

12.6

10.2

 

N

12

12

12

12

* / **: Dunnett-Test based on pooled variance significant at 5% (*) or 1% (**) level

Table 5. BODY WEIGHTS (GRAM) OF MALES SUMMARY P GENERATION - PAIRING

 

 

Group 1

0 ppm

Group 2

2500 ppm

Group 3

5000 ppm

Group 4

10000 ppm

 

Day 4

Mean

376

380

369

345**

 

ST. Dev

13.7

16.9

16.7

10.6

 

N

12

12

12

12

 

Day 8

Mean

391

393

384

359**

 

ST. Dev

15.2

17.5

14.6

12.5

 

N

12

12

12

12

 

Day 15

Mean

408

408

399

375**

 

ST. Dev

17.1

18.8

18.6

14.0

 

N

12

12

12

12

* / **: T-Test based on pooled variance significant at 5% (*) or 1% (**) level

Table 6. BODY WEIGHTS (GRAM) OF MALES SUMMARY P GENERATION - POST-PAIRING

 

 

Group 1

0 ppm

Group 2

2500 ppm

Group 3

5000 ppm

Group 4

10000 ppm

 

Day 1

Mean

411

411

401

377**

 

ST. Dev

19.1

18.8

19.0

14.4

 

N

12

12

12

12

 

Day 4

Mean

416

416

406

382**

 

ST. Dev

17.4

18.1

19.3

15.4

 

N

12

12

12

12

 

Day 7

Mean

422

425

412

387**

 

ST. Dev

18.2

20.3

17.2

16.7

 

N

12

12

12

12

 

Day 10

Mean

425

429

414

389**

 

ST. Dev

15.8

21.7

18.5

14.9

 

N

12

12

12

12

 

Day 12

Mean

423

427

415

390**

 

ST. Dev

15.5

23.1

18.2

14.9

 

N

12

12

12

12

* / **: Dunnett-Test based on pooled variance significant at 5% (*) or 1% (**) level

Table 7. BODY WEIGHTS (GRAM) OF FEMALES SUMMARY P GENERATION - PRE-PAIRING PERIOD

 

 

Group 1

0 ppm

Group 2

2500 ppm

Group 3

5000 ppm

Group 4

10000 ppm

 

Day 1

Mean

196

198

200

199

 

ST. Dev

11.3

9.1

9.6

9.7

 

N

12

12

12

12

 

Day 4

Mean

199

204

201

187*

 

ST. Dev

11.8

9.9

11.7

7.1

 

N

12

12

12

12

 

Day 7

Mean

204

208

205

192*

 

ST. Dev

12.1

10.5

12.1

9.5

 

N

12

12

12

12

 

Day 10

Mean

208

213

209

195*

 

ST. Dev

12.4

11.3

10.6

14.7

 

N

12

12

12

12

 

Day 13

Mean

214

219

216

203

 

ST. Dev

12.2

11.4

12.3

10.8

 

N

12

12

12

12

* / **: Dunnett-Test based on pooled variance significant at 5% (*) or 1% (**) level

Table 8. BODY WEIGHTS (GRAM) OF FEMALES SUMMARY P GENERATION - GESTATION

 

 

Group 1

0 ppm

Group 2

2500 ppm

Group 3

5000 ppm

Group 4

10000 ppm

 

Day 0

Mean

218

233*

218

208

 

ST. Dev

14.0

16.6

14.2

11.9

 

N

12

12

12

11

 

Day 7

Mean

243

255

244

233

 

ST. Dev

15.7

15.0

15.6

10.7

 

N

12

12

12

11

 

Day 14

Mean

273

283

272

252*

 

ST. Dev

20.1

15.9

16.8

12.2

 

N

12

12

12

11

 

Day 20

Mean

338

345

327

299**

 

ST. Dev

26.5

20.8

24.7

18.3

 

N

12

12

12

11

* / **: Dunnett-Test based on pooled variance significant at 5% (*) or 1% (**) level

Table 9. BODY WEIGHTS (GRAM) OF FEMALES SUMMARY P GENERATION - LACTATION

 

 

Group 1

0 ppm

Group 2

2500 ppm

Group 3

5000 ppm

Group 4

10000 ppm

 

Day 1

Mean

254

247

240

226**

 

ST. Dev

24.6

19.4

16.1

16.1

 

N

12

12

12

11

 

Day 4

Mean

270

266

263

233**

 

ST. Dev

20.5

23.2

14.1

14.0

 

N

12

12

12

11

* / ** : Dunnett-Test based on pooled variance significant at 5% (*) or 1% (**) level

Table 10. REPRODUCTION and BREEDING DATA – P GENERATION

 

 

Group 1

0 ppm

Group 2

2500 ppm

Group 3

5000 ppm

Group 4

10000 ppm

 

Litters

Total

12

12

12

11

 

Excluded

-

-

-

-

 

Evaluated

12

12

12

11

 

Duration of Gestation

Mean (+)

21.4

21.6 -

21.7 -

21.2 -

 

ST. Dev

0.5

0.5

0.5

0.4

 

N

12

12

12

11

 

Corpora Lutea

Total

186

179

170

137

 

Mean (+)

15.5

14.9 -

14.2 -

12.5 +

 

ST. Dev

2.6

1.6

2.8

1.9

 

N

12

12

12

11

 

Implantations

Total

151

164

145

122

 

Mean (+)

12.6

13.7 -

12.1 -

11.1 -

 

ST. Dev

1.8

1.8

2.2

2.2

 

N

12

12

12

11

 

Post Implantation Loss

% of Implantations

5.3

15.2

9.7

9.0

 

Litter affected (#)

4

6 -

9 -

7 -

 

Total (#)

8

25 ##

14 -

11 -

 

Mean (+)

0.7

2.1 -

1.2 -

1.0 -

 

ST. Dev

1.4

4.2

0.9

1.0

 

N

12

12

12

11

 

Dead Pups at First Litter Check

Litters affected (#)

-

1 -

-      -

2 -

 

Total (#)

0

14

0

3

 

Mean (+)

0.0

1.2 -

0.0 -

0.3 -

 

ST. Dev

0.0

4.0

0.0

0.6

 

N

12

12

12

11

#/##/- : Fisher's Exact Test, significant at 5% (#), 1% (##) or not significant (-)

+/++/-: Steel Test, significant at 5% (+), 1% (++) or not significant (-)

Applicant's summary and conclusion

Conclusions:
The NOEL (No Observed Effect Level) was established at the concentration level of 2500 ppm and the NOAEL (No Observed Adverse Effect Level) was established at 5000 ppm for general toxicity. For reproductive and developmental toxicity, the NOEL was established at 5000 ppm whereas the NOAEL was established at the concentration level of 10000 ppm, the highest concentration tested in the study.
Executive summary:

In a key combined repeated dose, reproductive/developmental toxicity screening study, the test material (Tall oil rosin, CAS# 8050-09-7) was administered daily in dietary mixtures at concentrations of 0, 2500 ppm (males - 160 mg/Kg bw/day Pre-Pairing and 136.4 mg/Kg bw/day Post-Pairing; females - 189 mg/Kg bw/day Pre-Pairing; 182.8 mg/Kg bw/day Gestation; and 241.1 mg/K bw/day Lactation), 5000 ppm (males - 301.5 mg/Kg bw/day Pre-Pairing and 276.5mg/K bw/day Post-Pairing; females – 356.1 mg/Kg bw/day Pre-Pairing; 377.3 mg/Kg bw/day Gestation; and 572.7 mg/K bw/day Lactation), or 10000 ppm (males - 496.4 mg/Kg bw/day Pre-Pairing and 585.5 mg/K bw/day Post-Pairing; females – 612.9 mg/Kg bw/day Pre-Pairing; 772.2 mg/Kg bw/day Gestation; and 1227.9 mg/K bw/day Lactation) to male rats for 42 days and to female rats for 14 days prior to pairing, through the pairing and gestation periods until the F1 generation reached day 4 post partum.

 

Treatment resulted in slightly higher test item intake in females compared to males during the pre-pairing period. Intake remained fairly constant throughout the entire study, except during the lactation phase when, due to an increase in food consumption, intake in females was almost two-fold higher than during the preceding gestation period. In the high concentration group (10000 ppm), a reduction in food consumption resulting in body weight loss followed by reduced body weight gain, significantly reduced body weights, and significantly reduced food conversion efficiency were observed in male and female rats. A recovery was observed for food consumption in males and females and for body weight gain and food conversion efficiency in males. In females, body weight gain and food conversion efficiency recovered also initially but both values reduced again during the gestation period and remained lower than the control values during the lactation period. Body weights in both males and females, remained reduced compared to the controls during the entire study. Body weight loss in one female led to premature termination of the animal for humane reasons. In females, effects on water consumption were also noted in the 10000 ppm group; an increase was noted during the gestation period followed by a reduction during the lactation period. Due to the recovery observed during the study and because Tall Oil Rosin (CAS # 8050-09-7) is known to cause palatability problems, effects on food consumption were considered to most probably not be a sign of toxic potential but resulted from unpalatability of the test item. The lower body weights in males of the 10000 ppm group compared to concurrent control throughout the study was considered to represent impaired growth resulting from food aversion during the early

treatment period. Similarly, lower body weights in females were considered to result from undernutrition at the beginning of the study. In females, however, a second drop in body weight gain and food consumption efficiency was observed during the gestation period after the recovery of food consumption. These later effects were considered to be related to potential toxicity of the test item. Reduction in body weights in both genders and reductions in body

weight gain and food conversion efficiency in females at the high concentration level were considered to be adverse because they were not reversible during the course of the study.

 

Further, terminal examinations revealed increased liver weights in males and females in the 10000 ppm group. This effect was accompanied by an increase in alkaline phosphatase activity but no changes in the blood activity of other liver enzymes were observed and there were no histopathological lesions in this organ in any gender. For these reasons, increased liver weights were considered not to be adverse and probably a result of increased metabolism due to the exposure to foreign compound.

 

In the high concentration group, the number of corpora lutea was reduced, which resulted in slightly, not statistically significant lower number of implantation sites and slightly, not statistically significant lower litter size at first litter check. Mean litter size at the high concentration level remained in the range of historical control values. Slightly reduced body weight and body weight gain were observed in pups during lactation. Although the reduction was only minor and predominantly not statistically significant if compared to the current control, values at the high concentration level were lower if compared to the historical control background and therefore this observation was considered to

be related to the treatment with the test item.

 

Mating performance, fertility, duration of gestation, post-implantation and post-natal loss were considered not to be affected by the treatment with the test item at any concentration level. Due to adverse effects on body weight observed in females in the high concentration group, a possibility that the reduced number of corpora lutea as well as lower pup body weight were secondary to the maternal response, should be taken into consideration.

 

In the intermediate concentration group (5000 ppm), a slight reduction in food consumption was observed in both genders at the beginning of the treatment. In males, this resulted in slight reductions in body weight gain and food conversion efficiency at the beginning of the treatment and slightly, not statistically significant, lower body weights during the entire study. In females, body weight gain and food conversion efficiency were also slightly reduced at the beginning of the study and recovered thereafter whereas body weights were not changed compared to the control values during the entire study. Food conversion efficiency reduced again during the gestation period but recovered and was higher than the control value during the lactation period. Water consumption was slightly and reversibly increased in females of this group. Due to reversibility, effects on food consumption, body weights and water consumption in the intermediate concentration group were considered not to be adverse. Although food conversion efficiency was significantly lower in this group again during the gestation period, it recovered during the lactation period and did not correlate with reduced body weight gain or reduced body weights and therefore this effect was also considered not to be adverse.

 

In the 5000 ppm group, increased liver weights were observed in males and females but without histopathological lesions or changes in related clinical biochemistry parameters and therefore considered not to be adverse. No effects on reproductive or developmental parameters were observed in animals in the 5000 ppm group.

 

In the low concentration group (2500 ppm), no signs of general toxicity in males or females as well as no effects on reproduction or development were observed.

 

Based on these observations, the NOEL (No Observed Effect Level) was established at the concentration level of 2500 ppm and the NOAEL (No Observed Adverse Effect Level) was established at 5000 ppm for general toxicity. For reproductive and developmental toxicity, the NOEL was established at 5000 ppm whereas the NOAEL was established at the concentration level of 10000 ppm, the highest concentration tested in the study.