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
Study period:
2013-10-15 to 2016-08-11
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-reference
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
Study period:
2013-10-15 to 2016-08-11
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
Clinical signs:
no effects observed
Description (incidence and severity):
There were no clinical signs apparent for those animals that survived to scheduled necropsy at 3000, 7500 and 18000/12000 ppm.
Mortality:
mortality observed, treatment-related
Description (incidence):
There were three unscheduled and treatment-related deaths on the study, all occurring at the highest dietary concentrations.

Male number 76, receiving diet contraining 18000 ppm, was killed on day 22 of the study after showing marked body weight loss, piloerection, emaciation and dehydration. Necropsy revealed increased pelvic space in the left kidney, dark liver, reddened lungs and small seminal vesicles.

Male number 78, receiving diet containing 18000 ppm, was also killed on day 22 of the study after showing marked body weight loss, piloerection, emaciation and diuresis. Necropsy revealed reddened lungs and small seminal vesicles.

Female number 96 receiving diet containing 18000/12000 ppm showed hunched posture and piloerection on day 24 and 25 of the study. On day 25, these signs were also accompanied by dehydration and the animal was killed and subjected to macroscopic necropsy. This revealed an increased pelvic space in a pale left kidney, thickened stomach, and dark foci and sloughing of the glandular region of the stomach. While this death occurred when the animal was receiving diet containing 12000 ppm, it is considered that these signs reflected the previous exposure to diet containing 18000 ppm.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Males:
Males at the 18000 ppm dietary level, showed notable mean body weight loss during the first week of dietary exposure, with differences attaining statistical significance. While recovery of body weight gain was apparent during the following week, body weight gain still remained statistically significantly lower than control. During Week 3, a number of individual animals showed marked body weight loss, resulting in a notable mean body weight loss at this dosage, although differences were not statistically significant when compared to control. The extent of the individual body weight losses observed was sufficient to trigger a decision to reduce the dietary dosage for high dietary level animals. Following the reduction in dietary concentration, no further mean body weight losses or statistically significant differences in body weight gain were apparent although overall body weight gain at the end of the study for these animals remained notably lower than control.

Body weight gains for males receiving diet containing 7500 ppm were generally lower than control throughout the study and although differenceswere only statistically significant on one occasion (days 22-29), statistically significant lower overall body weight gain was also apparent at the end of the study for these animals.

Body weight gains for males receiving diet containing 3000 ppm were also generally lower than control throughout the study and lower overall body weight gain was also apparent at the end of the study, however differences from control showed no statistical significance. Animals at the age used on this study are no longer on a steep growth curve and the differences observed were considered most likely to represent normal biological variation and reflect particularly good gain for the control males.

Females:
Females at the 18000 ppm dietary level, showed statistically significant mean body weight loss during the first week of dietary exposure. Recovery of body weight gain was apparent during the following week, with body weight gain slightly superior to control, although differences did not attain statistical significance. The high dietary concentration for females was scheduled to be reduced to 15000 ppm during gestation and lactation but was further reduced due to concerns regarding the body weight performance for some individual animals. Body weight gain of females at the 18000/12000 ppm dietary level was lower than control throughout gestation, with differences being most noticeable during the last week of gestation. Body weight gain during the last week of gestation is increasingly influencedby the weight of the developing litter and the differences from control in body weight gain observed during this period was, at least in part, influenced by smaller litter size for these animals in comparison to their control counterparts. A marginal mean body weight loss during lactation was apparent for females at the 18000/ 12000 ppm dietary level, compared to a gain in the control group, despite the lower litter size for the treated animals.

For females receiving the 7500 ppm dietary level, body weight gains during the two week prepairing period were lower than control but differences failed to attain statistical significance. Body weight gains were also slightly lower than control throughout gestation and lactation, with statistically significant differences during the second week of gestation and also during lactation.

For females receiving the 3000 ppm dietary level, body weight gains during the two week prepairing period and during gestation and lactation were considered to be unaffected by dietary exposure to the test item.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Males:
Males at the 18000 ppm dietary level, showed notably lower mean food consumption compared with control, during the first week of dietary exposure. While some recovery of food intake was apparent during Week 2, food consumption remained noticeably lower than control. Following the pairing period, food consumption was still noticeably lower than control, despite the reduction in dietary concentration to 12000 ppm at this stage of the study.

For males at the 7500 ppm dietary level, food consumption was similar to control during the prepairing phase of the study. However, food intake was slightly lower than control during the post-pairing period.

Food consumption of males at the 3000 ppm dietary level was considered to have been unaffected by dietary exposure to the test item throughout the study.

Females:
Food consumption of females at all dietary levelswas considered to have been unaffected by dietary exposure during the pre-pairing phase of the study. Food consumption of females at the 18000/12000 ppm dietary level was statistically significantly lower than control throughout gestation and lactation. The differences were particularly noticeable during the late gestation and the lactation phases of the study and may have been influenced by reduced demand on the dams resulting from a lower litter size.

Food consumption of females at the 7500 ppm dietary level was lower than control throughout gestation, but differences were not marked and only attained statistical significance during the second week of gestation. However food intakeduring lactation was clearly lower than control with differences attaining statistical significance.

Food consumption of females at the 3000 ppm dietary level was considered to have been unaffected by dietary exposure during the gestation and lactation phases of the study.
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
At the 18000 ppm dietary level, food conversion efficiency was lower for both sexes during the first week of dietary exposure (a group mean body weight loss was observed for both sexes). There was no obvious effect on food utilisation of either sex during the remaining pre-pairing phase or for males during the post-pairing phase of the study.

At the 3000 and 7500 ppm dietary level, food conversion efficiency appeared unaffected by dietary exposure of either sex during the pre-pairing phase or for males during the post-pairing phase of the study.
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
Males at the 18000 ppm dietary level, showed lower mean water consumption compared with control, throughout the two week pre-pairing period. This may be related to the lower food consumption observed for these animals during this period.

For males at the 7500 ppm dietary level, water intake during the pre-pairing period appeared lower than control from day 11 of the study. Prior to this, values did not indicate any consistent effect of dietary exposure to the test item.

For males at the 3000 ppm and females at all dietary levels, water consumption during the prepairing period was considered to have been unaffected by dietary exposure to the test item.
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
At the 18000/12000 ppm exposure level, males showed statistically significant lower mean corpuscular haemoglobin and females statistically significant lower mean cell haemoglobin concentration compared with control; however differences for these treated animals were all within the historical control range.

Females at all dietary levels, had statistically significant lower neutrophil counts compared with the control group, with means of 2.500, 1.104*, 1.258** and 0.0988* for control, low, intermediate and high dietary groups respectively. Mean values showed no consistent relationship to dietary exposure levels and among the test-item exposed animals only one individual value at 3000 ppm was below the historical control range (0.45-2.84) while two control values exceeded this historical range. It is considered that the observed differences reflect high control values rather than any effect of treatment.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
For females at the 7500 and 18000/12000 ppm dietary levels, total protein, albumin levels and albumin/globulin ratio were lower than control with differences attaining statistical significance. Mean values for both protein and albumin showed a consistent relationship to dietary exposure. The majority of individual values for these parameters were within the respective historical control ranges; one albumin value at the 18000/12000 ppm dietary level and one albumin/globulin ratio value at the 7500 ppm dietary level were outside their respective ranges and one control albumin value exceeded the historical range. Statistically significantly lower total protein and albumin levels were also observed for males at the 18000/ 12000 ppm dietary level but there was no accompanying statistically significantly effect on albumin/globulin ratio; only one individual value for total protein was below the historical control range for these treated animals. For males at all dosages, higher levels of potassium attained statistical significance compared to control; only two individual values at both the 3000 and 7500 ppm dietary level and one value at the 18000/ 12000 ppm dietary level exceeded the historical control range.

For both sexes at all dietary concentrations, higherlevels of total billirubin attained statistical significance when compared with control, with mean values showing a consistent dosage relationship; the majority of individual values at the 7500 and 18000/12000 ppm dietary levels exceeded the historical control range.

For females at the 7500 ppm dietary level and both sexes at the 18000/12000 ppm dietary level, alkaline phosphatase was lower than control with differences attaining statistical significance; the majority of individual values for these treated females were outside the historical control range but all values for these treated males were within the historical control range.

For females at the 18000/12000 ppm dietary level, lower inorganic phosphorus level attained statistical significance compared to control. However, as all individual values for these treated animals were within the historical control range and two control values exceeded this historical range, this finding was considered unlikely to be related to treatment and of no toxicological significance.

For females at the 7500 ppm dietary level, lower sodium levels also attained statistical significance compared to control; however only one individual value for these treated animals was outside the historical control range. In the absence of any similar findings at 18000/12000 ppm, this finding was considered to be incidental and unlikely to be related to treatment.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Behavioral Assessments
There were no treatment-related changes in the behavioural parameters at dietary concentrations of 3000, 7500 or 18000/12000 ppm.

One female at 3000 ppm dietary level showed palprebral closure on day 4 post partumbut this isolated finding, in the absence of any effects at higher dietary levels, was considered to be incidental and unrelated to treatment.

Functional Performance Tests
There were no changes in functional performance considered to be related to dietary exposure at 3000, 7500 or 18000/12000 ppm.

Sensory Reactivity Assessments
There were no inter-group differences in sensory reactivity scores that were considered to be related to dietary exposure at 3000, 7500 or 18000/12000 ppm.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
The test-item-related effects occurred with a greater incidence and/or severity than in controls or were present in treated animals but not in controls. They can be summarized as follows:

Adrenal Cortex:
- Minimal or mild diffuse hypertrophy of the zona glomerulosa in both sexes given diets containing 7500 or 18000/12000 ppm.
- Minimal diffuse atrophy of the zona fasciculata observed in 3/12 males given diets containing 7500 ppm and most animals of either sex given diets containing 18000/ 12000 ppm.

Bone Marrow:
- Minimal or mild increase in adipose tissue in one or more of the bone marrow samples examined from each animal in both sexes given diets containing 18000/12000 ppm and males given diets containing 7500 ppm.

Kidneys:
- Mild or moderate foci of basophilic tubules in 2/12 males and 1/12 females given diets containing 18000/12000 ppm.
- Minimal or mild single cell death in the affected tubules of these animals.
- Minimal to moderate pelvic dilatation, unilateral or bilateral, in both sexes given diets containing 18000/12000 and males given diets containing 7500 ppm.
- Mild hypertrophy of the collecting ducts in the papilla was seen in 2/12 males and 3/12 females given diets containing 18000/12000 ppm.

Liver:
- Minimal diffuse atrophy of hepatocytes in all the animals given diets containing 18000/12000 ppm and in 7/12 males and 9/12 females given diets containing 7500 ppm.

Mesenteric Lymph Node:
- Minimal to moderate intrasinusal congestion/erythrophagocytosis in both sexes given diets containing 18000/12000 ppm and males given diets containing 7500 ppm.

Pituitary Gland:
- Minimal diffuse atrophy of pituicytes in 7/11 females given diets containing 7500 and 10/12 females given diets containing 18000/12000 ppm.

Secondary Sex Glands of Males:
- Minimal or mild reduction in secretion was observed in the seminal vesicles of 5/12 males given diets containing 18000/12000 ppm one of which also had minimal reduced secretion in the prostate gland.

Spleen:
- Extramedullary haematopoiesis was lower in females only given diets containing 7500 or 18000/12000 ppm than in controls.

Thymus:
- Minimal or mild decreased cortical lymphocytes in both sexes given diets containing 18000/12000 ppm and females given diets containing 7500 ppm.

Urinary Bladder:
- Minimal to moderate diffuse hyperplasia of the urothelium in females given diets containing 7500 ppm and both sexes given diets containing 18000/12000 ppm.
- Associated minimal to moderate chronic active inflammation in most females given diets containing 7500 ppm and most females and some males given diets containing 18000/ 20000 ppm.
- In a few affected animals of the highest dietary exposure group erosion or ulceration of the mucosa.
- In a female from the highest dietary exposure group, minimal focal submucosal haemorrhage.
- Minimal inflammatory cell infiltration of the submucosal in 2/7 females given diets containing 7500 ppm.

The test item-related changes in urinary bladder and kidneys were considered adverse and due to an irritant effect of the compound or most likely, a metabolite to the urothelium. More severe effects occurring in the urinary bladder probably because of the longer time that the mucosa is exposed to the irritant.

With the exception of the change seen in the mesenteric lymph node the test-item-related effects in the other tissues could all be the result ofcomparative differences in food consumption and body weight gain and, in the zona glomerulosa, the effects of dehydration on electrolyte imbalance, Levin et al(1993).

The change seen in the mesenteric lymph nodes was unexplained but was considered of minor biological importance and therefore non-adverse.

All of the other histopathological findings encountered were considered to have occurred spontaneously or post mortem. In addition to the routine evaluation of the testes, the seminiferous tubules of the testes were evaluated with respect to their stage in the spermatogenic cycle and the integrity of the various cell types present within the different stages. No treatment related abnormalities were noted.
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Fertility: Fertility as assessed by pregnancy rate was unaffected by dietary exposure at all dietary levels.

Gestation Length: Gestation lengths (where mating was observed) were between 22 and 23½ days and the distribution of gestation lengths for treated females was essentially similar to control and did not indicate any effect of dietary exposure.

At the 18000/12000 ppm dietary level, mean corpora lutea number was lower than control, with differences attaining statistical significance. Although subsequent pre and post implantation losses were similar to control, mean implantation count and litter size at birth/post-natal day 1 remained statistically significantly lower than control. One female (No. 85) showed total litter loss post partum, however post natal survival for the remaining litters was similar to control, although litter size continued to be significantly lower than control to post-natal day 4 of age. Sex ratio was unaffected indicating that maternal dietary exposure produced no selective effect on the survival of the offspring at this dietary level.

There was considered to be no obvious effect ofdietary exposure on the numbers of corpora lutea, implantations, pre and post implantation losses, litter size at birth/post-natal day 1 and post natal day 4 of age and post natal survival at the 3000 or 7500 ppm dietary levels. At 3000 ppm, lower corpora lutea count attained statistical significance when compared to control but this was considered not to represent an effect of treatment, as no statistically significant effect was apparent for corpora lutea count at 7500 ppm.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
In addition to the routine evaluation of the testes, the seminiferous tubules of the testes were evaluated with respect to their stage in the spermatogenic cycle and the integrity of the various cell types present within the different stages. No treatment related abnormalities were noted.
Reproductive performance:
effects observed, non-treatment-related
Description (incidence and severity):
Mating:
There was no obvious effect of dietary exposure on mating performance with the majority of matings occurring within the first four days of pairing (i.e. at the first estrus opportunity).

As previously discussed in the mortality section, female 96 (receiving 18000/12000 ppm) was killed in extremisduring the pairing period (study day 25). This animal did not show any evidence of mating prior to being terminated, however this was considered not to indicate any effect on mating performance at this dietary level.
Key result
Dose descriptor:
NOAEL
Effect level:
ca. 3 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Systemic Toxicity
Remarks on result:
other: Mean Achieved Dosage Level equivalent to 179.3 mg/kg bw/day in males and 221.5 mg/kg bw/day in females
Key result
Dose descriptor:
NOAEL
Effect level:
ca. 7 500 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Reproductive Toxicity
Remarks on result:
other: Mean Achieved Dosage Level equivalent to 441.9 mg/kg bw/day in males and 529.7 mg/kg bw/day in females
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
7 500 ppm
System:
urinary
Organ:
bladder
kidney
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
The clinical signs apparent for offspring on the study were typical for the age observed and did not indicate any clear effect of maternal dietary exposure on offspring development at 3000, 7500 and 18000/12000 ppm.

At 7500 and 18000/12000 ppm there was a slightly higher incidence of clinical signs for the offspring however, these tended to be concentrated in only a few litters, and overall did not indicate a clear effect of maternal dietary exposure on offspring development. These findings included no milk visable in stomach (2 and 3 litters at 7500 ppm and 18000/12000 ppm respectively), cold (1 litter at 18000/12000 ppm), weak (1 litter at 18000/12000 ppm), small (2 and 3 litters at 7500 ppm and 18000/12000 ppm respectively) and distended abdomen (1 litter at 7500 ppm).
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
In total 12, 12, 12 and 10 females from the control, 3000, 7500 and 18000/12000 ppm dietary groups gave birth to a live litter and successfully reared young to post-natal day 4. Additionally, one female at the 18000/12000 ppm dietary level showed total litter loss post partum.

At the 18000/12000 ppm dietary level one female (No. 85) showed total litter loss post partum, however post natal survival for the remaining litters was similar to control, although litter size continued to be significantly lower than control to post-natal day 4 of age. There was considered to be no obvious effect of dietary exposure on litter size at birth/post-natal day 1 and post natal day 4 of age and post natal survival at the 3000 or 7500 ppm dietary levels.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At the 18000/12000 ppm dietary level, offspring body weight on days 1 and 4 and subsequent body weight gain to day 4 was lower than concurrentcontrol, despite the lower litter size at this dietary level, with differences attaining statistical significance. Litter weights were also statistically significantly lower than control,reflecting both lower pup body weights and the smaller litter size.

At the 7500 ppm dietary level, offspring body weight on post-natal day 1 and 4 post partumand subsequent body weight gain to post-natal day 4 were also lower than concurrent control, with differences attaining statistical significance. Lower litter weight also attained statistical significance on post-natal day 4 of age.

At the 3000 ppm dietary level, pup body weight on post-natal day 1 and subsequent pup body weight gain to post-natal day 4 of age and litter weights at post-natal day 1 and 4 were considered to be unaffected by maternal dietary exposure.
Sexual maturation:
no effects observed
Description (incidence and severity):
Sex ratio was unaffected indicating that maternal dietary exposure produced no selective effect on the survival of the offspring at all dietary levels.
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
At the 7500 and 18000/12000 ppm dietary level, offspring performance during assessment of surface righting was inferior to control, although the mean values did not show a relationship to dietary exposure level. Additionally, in the high exposure group, all of the offspring from the one litter that died before scheduled termination on day 5 failed to showed righting reflex.

At the 3000 ppm dietary level, surface righting ability of the offspring appeared to be unaffected by maternal dietary exposure to the test item.
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
ca. 3 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Survival, growth and development
Remarks on result:
other: Mean Achieved Dosage Level equivalent to 179.3 mg/kg bw/day in males and 221.5 mg/kg bw/day in females
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
yes
Lowest effective dose / conc.:
7 500 ppm
Treatment related:
yes
Relation to other toxic effects:
not specified
Dose response relationship:
yes
Relevant for humans:
not specified

Table 7. Group Mean Body Weight Values - Females (P0)

 

Group

 

Day Numbers

 

Gestation

Lactation

 

0

7

14

20

1

4

Control

Mean

226.2

255.8

284.3

347.2

262.8

277.1

S.D.

9.3

9.6

11.5

21.0

10.8

8.6

N

12

12

12

12

12

12

 

Low Concentration

Mean

227.3

253.5

281.9

341.5

261.2

269.7

S.D.

7.8

9.5

12.5

18.6

11.8

10.7

N

11

11

11

11

12

12

 

Intermediate

Concentration

Mean

217.0*

244.3*

266.6**

319.9**

244.0**

244.6***

S.D.

9.6

12.4

13,0

21.0

13.4

13.9

N

12

12

12

12

12

12

 

High Concentration

Mean

206.8***

227.4***

245.2***

275.0***

219.8***

218.8***

S.D.

9.0

9.1

12.0

9.6

10.1

10.7

N

10

10

10

10

10

10

Table 8. Group Mean Body Weight Gains – P0 - Males (Increase in Body Weight (g))

 

 

Day Numbers Relative to Start Date

 

 

 

 

 

 

 

 

Abs Gain

% Gain

Group

From

1

8

15

22

29

36

1

1

To

8

15

22

29

36

43

43

43

 

Control

Mean

27.2

21.3

7.8

18.1

16.8

7.2

98.3

30.2

S.D.

6.8

5.5

5.0

7.3

7.2

6.9

25.1

7.2

N

12

12

12

12

12

12

12

12

 

Low Concentration

Mean

21.6

18.2

8.1

11.6

13.0

4.9

77.3

24.1

S.D.

9.5

7.8

7.5

5.1

3.9

6.7

25.2

8.0

N

12

12

12

12

12

12

12

12

 

Intermediate

Concentration

Mean

21.8

17.0

5.3

10.1*

14.8

5.5

74.3*

23.1

S.D.

6.4

6.1

6.8

6.2

6.5

4.9

25.8

7.7

N

12

12

12

12

12

12

12

12

 

High Concentration

Mean

-6.9**

12.3**

-12.6

21.4

15.3

4.9

47.9***

15.0

S.D.

9.9

5.4

29.5

11.3

4.3

3.4

14.8

4.4

N

12

12

12

10

10

10

10

10

Table 9. Group Mean Body Weight Gains – P0 - Females

 

 

 

Increase in Body Weight (g)

Cumulative Body Weight Change (g)

Days

 

Group

 

Gestation

Lactation

Gestation

From

0

7

14

1

0

0

To

7

14

20

4

14

20

 

Control

Mean

29.7

28.4

62.9

14.3

58.1

121.0

S.D.

5.2

7.2

16.0

6.8

9.7

19.6

N

12

12

12

12

12

12

 

Low Concentration

Mean

26.3

28.4

59.6

8.5

54.6

114.3

S.D.

5.3

4.6

7.5

4.9

8.1

14.0

N

11

11

11

12

11

11

 

Intermediate

Concentration

Mean

27.3

22.3*

53.3

0.6***

49.6

102.9*

S.D.

7.3

6.0

9.2

8.5

9.7

16.7

N

12

12

12

12

12

12

 

High Concentration

Mean

20.6**

17.8***

29.8***

-1.0***

38.4***

68.2***

S.D.

6.8

4.4

7.5

5.8

9.1

11.5

N

10

10

10

10

10

10

Table 10. Group Mean Food Consumptions – P0 - Females

 

 

Group

 

Day Numbers

 

Gestation

Lactation

From

0

7

14

1

To

7

14

20

4

Control

Mean

19.5

22.5

22.7

28.4

S.D.

1.3

2.1

1.5

3.8

N

12

12

12

12

Low Concentration

Mean

19.3

21.7

23.4

27.0

S.D.

1.5

2.0

2.0

6.1

N

11

11

11

12

Intermediate

Concentration

Mean

18.1

19.3**

21.7

19.9***

S.D.

2.1

2.1

1.6

2.8

N

12

12

12

12

High Concentration

Mean

17.0*

19.0**

17.7***

14.5***

S.D.

1.3

2.8

1.4

3.6

N

10

10

10

10

Table 11. Group Mean Litter Size and Litter Weights

 

 

 

 

Group

 

Number

of

Corpora

Lutea

Number of

Implantation

Sites

Total

Number of

Offspring

Born

Number of Live

Offspring

Litter Weight

(g)

Offspring Weight (g)

Offspring

Body Weight Change

(g)

 

 

 

 

 

Day 1

Day 4

Day 1

Day 4

Day 1

Day 4

Day 1

Day 4

Days 1-4

 

 

 

 

 

 

 

 

Males

females

Males

females

Males

females

 

Control

Mean

14.3

12.9

12.0

11.8

11.8

71.80

102.87

6.24

6.09

9.08

8.92

2.84

2.83

S.D.

2.1

3.6

3.3

3.3

3.2

18.5

24.39

0.55

0.55

1.16

1.15

0.66

0.61

N

12

12

12

12

12

12

12

12

12

12

12

12

12

 

Low Concentration

Mean

11.8*

11.8

11.2

11.2

11.0

67.43

96.67

6.33

5.95

9.12

8.80

2.79

2.84

S.D.

2.8

2.7

2.7

2.7

2.5

13.76

17.91

0.69

0.63

1.07

1.05

0.47

0.48

N

12

12

12

12

12

12

12

12

12

12

12

12

12

 

Intermediate

Concentration

Mean

12.4

12.0

11.2

11.2

10.8

60.95

77.34**

5.63*

5.37**

7.45**

7.06**

1.82***

1.70***

S.D.

2.4

2.3

2.5

2.5

2.2

12.33

15.75

0.54

0.44

1.06

1.01

0.64

0.61

N

12

12

12

12

12

12

12

12

12

12

12

12

12

 

High Concentration

Mean

9.0**

8.3***

7.6**

7.6**

7.4**

39.43***

49.29***

5.39**

5.16***

6.92***

6.68**

1.53***

1.52***

S.D.

2.5

1.7

1.4

1.4

1.5

6.61

8.21

0.65

0.60

1.18

1.08

0.63

0.65

N

10

10

10

10

10

10

10

10

10

10

10

10

10

Table 12. Group Mean Blood Chemical Values – P0 - Males

Group

 

Total Prot.(g/dL)

Albumin

(g/dL)

K+

(mmol/L)

AP

(IU/L)

Bili

(mg/dL)

 

Control

Mean

6.832

3.92

4.530

142.8

0.096

S.D.

0.197

0.08

0.179

17.6

0.005

N

5

5

5

5

5

 

Low Concentration

Mean

6.794

3.78

4.966*

141.6

0.114*

S.D.

0.226

0.18

0.581

27.4

0.005

N

5

5

5

5

5

 

Intermediate

Concentration

Mean

6.684

3.74

4.916*

149.8

0.192**

S.D.

0.208

0.21

0.184

56.6

0.026

N

5

5

5

5

5

 

High Concentration

Mean

6.378*

3.58**

5.150*

95.4**

0.332**

S.D.

0.502

0.16

1.068

14.2

0.045

N

5

5

5

5

5

Table 13. Group Mean Blood Chemical Values – P0 - Females

Group

 

Total Prot.

(g/dL)

Albumin

(g/dL)

A/G Ratio

Na+

(mmol/L)

P

(mmol/L)

AP

(IU/L)

Bili

(mg/dL)

 

Control

Mean

6.874

3.96

1.366

147.0

1.52

136.8

0.058

S.D.

0.391

0.27

0.051

1.4

0.55

33.1

0.011

N

5

5

5

5

5

5

5

 

Low Concentration

Mean

6.600

3.76

1.328

148.2

1.38

167.4

0.082*

S.D.

0.420

0.34

0.105

4.1

0.60

62.5

0.022

N

5

5

5

5

5

5

5

 

Intermediate

Concentration

Mean

6.272**

3.44**

1.218*

141.0*

1.02

71.0*

0.188**

S.D.

0.200

0.15

0.102

4.8

0.41

19.2

0.085

N

5

5

5

5

5

5

5

 

High Concentration

Mean

5.934**

3.36**

1.284*

143.6

0.82*

47.6**

0.396**

S.D.

0.311

0.18

0.044

2.3

0.26

35.4

0.247

N

5

5

5

5

5

5

5

Table 14. Group Mean Organ Weights with Corresponding Relative (% of Body Weight) Organ Weights

 

Organ

 

Males

Females

0

Control

3000

ppm

7500

ppm

18000 /

12000

ppm

0

Control

3000

ppm

7500

ppm

18000 /

12000

ppm

 

Liver

Mean (g)

13.6882

13.2353

13.2295

12.9473**

11.7482

12.3408**

9.96312

8.85520

S.D.

1.51014

1.30389

1.75721

1.46364

1.20970

1.38803

0.90712

0.59784

N

12

12

12

10

12

12

12

10

 

Mean (%)

3.254

3.312

3.327

3.525**

4.164

4.516**

4.023

4.007

S.D.

0.159

0.201

0.263

0.259

0.314

0.433

0.236

0.289

N

12

12

12

10

12

12

12

10

 

Ovaries

Mean (g)

 

 

 

 

0.12826

0.10733**

0.09420*

0.08598

S.D.

 

 

 

 

0.01814

0.01303

0.01810

0.01552

N

 

 

 

 

12

12

12

10

 

Mean (%)

 

 

 

 

0.046

0.039**

 

0.038*

0.039

S.D.

 

 

 

 

0.007

0.005

0.006

0.007

N

 

 

 

 

12

12

12

10

 

Pituitary

Mean (g)

0.01471

0.01258*

0.01234*

0.01077*

0.01759

0.01716

0.01464*

0.01310**

S.D.

0.00286

0.00309

0.00156

0.00145

0.00271

0.00178

0.00250

0.00116

N

12

12

12

10

12

12

12

10

 

Mean (%)

0.004

0.003*

0.003*

0.003**

0.006

0.006

0.006**

0.006**

S.D.

0.001

0.001

0.000

0.000

0.001

0.001

0.001

0.000

N

12

12

12

10

12

12

12

10

 

Seminal Vesicles

Mean (g)

2.26419

2.05758

1.89807**

1.56437**

 

 

 

 

S.D.

0.33826

0.40448

0.27070

0.30683

 

 

 

 

N

12

12

12

10

 

 

 

 

 

Mean (%)

0.543

0.516

0.479**

0.429**

 

 

 

 

S.D.

0.095

0.099

0.061

0.091

 

 

 

 

N

12

12

12

10

 

 

 

 

 

Spleen

Mean (g)

0.76190

0.78678

0.64450

0.80042

0.67432

0.59530**

0.59402*

0.46908**

S.D.

0.10345

0.04341

0.11716

0.08103

0.05867

0.05576

0.05440

0.02662

N

5

5

5

5

5

5

5

5

 

Mean (%)

0.178

0.190

0.167

0.214

0.238

0.216**

0.233*

0.209**

S.D.

0.020

0.016

0.026

0.025

0.028

0.026

0.020

0.015

N

5

5

5

5

5

5

5

5

 

Uterus and Cervix

Mean (g)

 

 

 

 

0.70715

0.65747

0.68088

0.55625**

S.D.

 

 

 

 

0.09892

0.12481

0.15165

0.10713

N

 

 

 

 

12

12

12

10

 

Mean (%)

 

 

 

 

0.251

0.240

0.274

0.251**

S.D.

 

 

 

 

0.038

0.039

0.055

0.045

N

 

 

 

 

12

12

12

10

Conclusions:
Based on the results this study the No Observed Adverse Effect Level (NOAEL) for systemic toxicity was considered to be 3000 ppm, principally due to effects on body weight gain, food consumption and adverse histopathological changes in the kidney and urinary bladder at 7500 ppm and 18000/12000 ppm.

The No Observed Adverse Effect Level (NOAEL)for reproduction was considered to be 7500 ppm due to lower corpora lutea count at 18000/12000 ppm.

The No Observed Adverse Effect Level (NOAEL) for survival, growth and development of the offspring was considered to be 3000 ppm due to effects on body weight, body weight gain and surface righting ability at 7500 ppm and 18000/12000 ppm.
Executive summary:

This data is being read across from the source study that tested Resin acids and Rosin acids, maleated, esters with pentaerythritol based on category read across that is explained in the category justification document attached in Section 13 of the dossier.

In a key combined repeat dose toxicity study with reproduction/developmental toxicity screening test, the test material (Resin acids and rosin acids, maleated, esters with pentaerythritol, CAS# 94581-17-6) was administered in the diet to three groups, each composed of twelve male and twelve female Wistar Han™:RccHan™:WIST strain rats, for up to eight weeks (including a two week pre-pairing phase, pairing, gestation and early lactation for females), at dietary concentrations of 3000, 7500 and 18000 ppm. The dietary concentration given to the high dietary concentration females during gestation and lactation was initially decreased to 15000 ppm to lessen the expected increase in achieved intake during these phases. However, due to adverse toxicity, the high dietary concentration was reduced to 12000 ppm for both sexes on study day 22. Estimated achieved dosages for males in Groups 2 to 4 during the study was 179.3, 441.9 and 783.4 mg/kg bw/day respectively. For females, it was 221.5, 529.7 and 1508.1 mg/kg bw/day during the pre-pairing phase, 237.0, 570.8 and 911.6 during gestation and 305.1, 610.9 and 794.0 mg/kg bw/day during lactation respectively. A control group of twelve males and twelve females were treated with basal laboratory diet.

 

Clinical signs, behavioural assessments, body weight change and food and water consumption were monitored during the study. Pairing of animals within each dose group was undertaken on a one male: one female basis within each treatment group on study day 15, with females subsequently being allowed to litter and rear their offspring to lactation day 5. During the lactation phase, daily clinical observations were performed on all surviving offspring, together with litter size and offspring weights and assessment of surface righting reflex. Extensive functional observations were performed on five males from each dose group after the completion of the pairing phase, and for five parental females from each dose group on day 4 post-partum.Haematology and blood chemistry were evaluated prior to termination on five males and females from each dose group. Surviving adult males were terminated on study day 43/44, followed by the termination of all surviving females and offspring on day 5 post-partum. All adult animals, including decedents were subjected to a gross necropsy examination and histopathological evaluation of selected tissues was performed.

 

There were three unscheduled and treatment-related deaths at the highest dietary level (18000 ppm). No signs of treatment-related clinical toxicity were apparent in any of the surviving animals at 3000, 7500 and 18000/ 12000 ppm. Behavioural assessments, functional performance, sensory reactivity assessments, and haematology parameters were unaffected by dietary exposure at 3000, 7500 and 18000/ 12000 ppm. Gross necropsy did not reveal any remarkable findings in rats of either sex.

 

At the 18000 ppm dietary concentration, notable mean body weight loss during the first week of dietary exposure and statistically significant lower body weight gain during Week 2, compared with control, was observed in males. During Week 3, a number of individual animals showed marked body weight loss, resulting in a mean bodyweight loss and the dietary concentration was lowered to 12000 ppm for the high dietary level group. No further mean body weight losses or statistically significant differences in body weight gain were apparent for the remainder of the study although the overall body weight gain at termination of the study remained statistically significantly lower than control. Females in the 18000 ppm group, showed notable mean body weight loss during the first week of dietary exposure but recovery of body weight gain was apparent during the following week. Body weight gain of females at 18000/12000 ppm was lower than control throughout gestation, particularly during the last week of gestation. During lactation, marginal mean body weight loss was apparent for females in the 18000/12000 ppm group compared with control. Body weight gains for males receiving diets containing 7500 ppm were generally lower than control throughout the study and statistically significant lower overall body weight gain was apparent for these animals at the end of the study. Body weight gains for females that received diets containing 7500 ppm were lower than control during the two week pre-pairing period and throughout gestation and lactation, although differences only attained statistical significance during the second week of gestation and during lactation. There was no obvious effect of dietary exposure to 3000 ppm on body weight gains for both sexes throughout the study.

 

At the 18000 ppm dietary concentration, males showed notably lower mean food consumption during the first and, to a lesser extent, second week of dietary exposure compared with control. Following the pairing period, food consumption remained lower than control, despite the animals only receiving 12000 ppm at this stage of the study. For females at the 18000/12000 ppm dietary concentration, food consumption was lower than control throughout gestation and lactation phases; these differences were particularly noticeable during late gestation and during lactation. At the 7500 ppm dietary concentration, food consumption for males was slightly lower than control during the post-pairing period. Food consumption for females was lower than control throughout gestation, and, to a greater extent, lactation. At the 3000 ppm dietary concentration, food consumption for each sex was considered to have been unaffected by dietary exposure to the test material.

 

At the 18000 ppm dietary concentration, food conversion efficiency was lower for both sexes during the first week of dietary exposure to the test item. At the 3000 and 7500 ppm dietary concentrations, food conversion efficiency for both sexes appeared to be unaffected by dietary exposure to the test material.

 

At the 18000 ppm dietary concentration, males showed lower mean water consumption compared with control, throughout the two week pre-pairing period. For males at the 7500 ppm dietary concentration, water intake during the pre-pairing period appeared lower than control from day 11 of the study; prior to this, values did not indicate any consistent effect of dietary exposure. For males at the 3000 ppm dietary concentration and females at all dietary levels, water consumption during the pre-pairing period was unaffected by dietary exposure.

 

For both sexes at all dietary concentrations, statistically significant higher levels of total bilirubin were observed when compared with control, with mean values showing a consistent relationship to test material exposure. The majority of individual values at the 7500 and 18000/12000 ppm dietary concentrations exceeded the historical control range. All individual values for the 3000 ppm dietary group were within the historical control range. For females at the 7500 and 18000/12000 ppm dietary concentrations, statistically significant lower total protein, albumin and albumin/globulin ratio were observed compared with control; however the majority of individual values were within the historical control range. Statistically significant lower total protein and albumin levels were also observed for males at the 18000/12000 ppm dietary concentrations, however, the majority of individual values were within the historical control range. For females at the 7500 ppm dietary level and both sexes at the 18000/12000 ppm dietary level, alkaline phosphatase was lower than control with differences attaining statistical significance; the majority of individual values for these exposed females were below the historical control range but all values for these exposed males were within the historical control range. For males at all dietary concentrations, statistically significant higher levels of potassium were observed compared to control, however, the majority of individual values were within the historical control range.

 

Although macroscopic necropsy findings at termination did not indicate any obvious effect of dietary exposure to the test material at 3000, 7500 and 18000/12000 ppm, a number of organ weights attained statistical significance compared to control: Males at the 18000/12000 ppm dietary level showed lower absolute liver weight and higher relative liver weights compared to control, although the majority of individual values were within the historical control range. Microscopic evaluation revealed minimal diffuse atrophy of hepatocytes for both sexes at 18000/12000 ppm and, to a lesser extent, at 7500 ppm.

 

Females at the 7500 and 18000/12000 ppm dietary levels, showed lower absolute and relative pituitary weight compared to control, although the majority of individual values were within the historical control range. Microscopic evaluation revealed minimal diffuse atrophy of pituicytes for females at the 18000/12000 ppm and, to a lesser extent, at the 7500 ppm dietary level. Males receiving diets containing 7500 ppm or 18000/12000 ppm of the test item showed lower absolute and relative seminal vesicles weights compared to control. At the 18000/12000 ppm exposure level a number of individual seminal vesicle values were outside the historical control range and microscopic evaluation revealed a minimal or mild reduction in secretion in the seminal vesicles. This finding at the high dietary exposure level was therefore considered to be treatment related. For males receiving 7500 ppm of the test material all individual seminal vesicles weights were within the historical control and in the absence of any supporting histopathological change, the decrease in seminal vesicle weight at this dietary concentration was considered not to be of toxicological significance. Females at all dietary levels showed lower absolute and body weight relative spleen weights compared with control but all individual absolute and body weight relative values for treated animals were within the historical control range. Microscopic evaluation revealed lower extramedullary haematopoiesis for females at the 7500 or 18000/12000 ppm dietary levels compared to control, therefore the lower spleen weights at these exposure levels were considered to be related to treatment. At the 3000 ppm exposure level, there was no evidence of histopathological change and the lower spleen weights were considered to be of no toxicological significance.

 

These differences in organ weight and accompanying microscopic changes were considered to reflect comparative differences in food consumption and body weight gain and, in the zona glomerulosa, the effects of dehydration on electrolyte imbalance, (Levin et al.,1993). Other microscopic changes also considered to be attributable to these differences included: Minimal or mild diffuse hypertrophy of the zona glomerulosa of the adrenal cortex in both sexes at the 7500 or 18000/12000 ppm dietary levels and minimal diffuse atrophy of the zona fasciculata of the adrenal cortex in both sexes at the 18000/12000 ppm dietary level and, to a lesser extent, males at the 7500 ppm dietary level.

 

Minimal or mild increase in adipose tissue was observed in the bone marrow samples of males at the 7500 ppm dietary level and in both sexes at the 18000/12000 ppm dietary level. Minimal reduced secretion in the prostate gland of one male was observed at the 18000/12000 ppm dietary level. Minimal or mild decreased cortical lymphocytes of the thymus was observed in females at the 7500 ppm dietary level and in both sexes at 18000/12000 ppm dietary level.

 

Other microscopic changes, considered to be more serious in nature, were also observed. For the kidney, mild or moderate foci of basophilic tubules were apparent for a few animals at the 18000/12000 ppm dietary level, these animals also showed minimal or mild single cell death in the affected tubules. Minimal to moderate pelvic dilatation was also apparent for males at the 7500 ppm dietary level and both sexes at the 18000/12000 ppm dietary level and mild hypertrophy of the collecting ducts in the papilla was seen a few animals at the 18000/ 12000 ppm dietary level. For the urinary bladder, minimal to moderate diffuse hyperplasia of the urothelium, with associated minimal to moderate chronic active inflammation, was apparent for females at the 7500 ppm dietary level and in both sexes at the 18000/12000 ppm dietary level. At the highest exposure level, a few affected animals also showed erosion or ulceration of the mucosa and one female showed minimal focal submucosal haemorrhage. Two females at the 7500 ppm dietary level also showed minimal inflammatory cell infiltration of the submucosa. The observed changes in the urinary bladder and kidneys were considered adverse and due to an irritant effect of the test item, or a metabolite, to the urothelium. The more severe effects apparent in the urinary bladder was probably due to the longer time that the urinary bladder mucosa was exposed to the test item or metabolite. Microscopic changes were also apparent in the mesenteric lymph nodes, with minimal to moderate intrasinusal congestion/erythrophagocytosis being observed in males at the 7500 ppm dietary level and in both sexes at the 18000/12000 ppm dietary level.

 

Mating performance, pregnancy rate, and gestation length were unaffected by dietary exposure to the test material. At the 18000/12000 ppm dietary concentration, the mean number of corpora lutea was lower than control, resulting in a lower number of implantations and litter size on post-natal days 1 and 4. However, no obvious effect on pre-natal and post-natal survival or sex ratio was observed at this exposure level. At the 3000 and 7500 ppm dietary concentrations, there were no obvious effects on numbers of corpora lutea, implantations, pre-and post implantation losses, litter size or sex ratio at birth/postnatal day 1 and post-natal day 4 of age or on post-natal pup survival.

 

At the 18000/12000 ppm dietary concentration, offspring bodyweight weight and litter weights on post-natal days 1 and 4 and offspring body weight gain to post-natal day 4 were lower than control. Offspring performance during assessment of surface righting on post-natal day 1 was also inferior to control at the highest exposure level. At the 7500 ppm dietary concentration, offspring bodyweight weight on post-natal days 1 and 4, offspring body weight gain to post-natal day 4 and litter weight on post-natal day 4 were lower than control. Offspring performance during assessment of surface righting was also inferior to control at the intermediate exposure level. At the 3000 ppm dietary concentration, offspring body weights, litter weights, offspring body weight gain and surface righting ability of the offspring were considered to be unaffected by maternal dietary exposure to the test material.

 

At the 7500 and 18000/12000 ppm dietary concentrations, there was a slightly higher incidence of clinical signs among offspring but these were concentrated in only a few litters (no milk visible in stomach (2 and 3 litters at 7500 ppm and 18000/12000 ppm respectively), cold (1 litter at 18000/12000 ppm), weak (1 litter at 18000/12000 ppm), small (2 and 3 litters at 7500 ppm and 18000/12000 ppm respectively) and distended abdomen (1 litter at 7500 ppm)), and did not indicate a clear effect of maternal dietary exposure on offspring development.

 

Based on the results this study the No Observed Adverse Effect Level (NOAEL) for systemic toxicity was considered to be 3000 ppm, principally due to effects on body weight gain, food consumption and adverse histopathological changes in the kidney and urinary bladder at 7500 ppm and 18000/12000 ppm.

 

The NOAEL for reproduction was considered to be 7500 ppm due to lower corpora lutea count at 18000/12000 ppm.

 

The NOAEL for survival, growth and development of the offspring was considered to be 3000 ppm due to effects on body weight, body weight gain and surface righting ability at 7500 ppm and 18000/12000 ppm.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

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:
Deviations were considered not to have affected the scientific integrity of the study.
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Han™:RccHan™:WIST
Details on species / strain selection:
The rat was selected for this study as it is a readily available rodent species historically used in safety evaluation studies and is acceptable to appropriate regulatory authorities.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories U.K. Ltd. (Blackthorn, Bicester, Oxon, UK)
- Females (if applicable) nulliparous and non-pregnant: not specified
- Age at study initiation: (P) x 12 wks; (F1) x wks
- Weight at study initiation: (P) Males: 296 to 346 g; Females:193 to 228 g; (F1) Males: x-x g; Females: x-x g
- Fasting period before study: not specified
- Housing: Initially, all animals were housed in groups of three in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK). During the pairing phase, animals were transferred to polypropylene grid floor cages suspended over trays lined with absorbent paper on a one male: one female basis within each dose group. Following evidence of successful mating, the males were returned to their original cages. Mated females were housed individually during gestation and lactation in solid floor polypropylene cages with stainless steel mesh lids and softwood flakes.
- Diet (e.g. ad libitum): Ground diet (Rodent PMI 5002 (Certified), BCM IPS Limited, London, UK) was provided ad libitum.
- Water (e.g. ad libitum): Mains drinking water was supplied ad libitum from polycarbonate bottles attached to the cage.
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 C
- Humidity (%): 50 ± 20%
- Air changes (per hr): at least fifteen air changes per hour
- Photoperiod (hrs dark / hrs light): low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness.

IN-LIFE DATES: From: 2013-101-5 To: 2013-12-06

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): Formulations were prepared approximately fortnightly
- Mixing appropriate amounts with (Type of food): For each dietary concentration an appropriate amount of test item and diet were weighed out. A suitable sub-sample of the test item was added to an appropriate subsample of the diet in a mixing container and shaken to initially mix the two ingredients and then transferred to a Robot Coupe Blixer 4 mixer. This procedure was repeated until all the test item had been used. These initial mixtures were thenmixed together until considered homogenous. The resulting pre-mix was then transferred to a Hobart H800 mixer and the remaining diet was added and mixed until considered homogenous.
- Storage temperature of food: stored under ambient conditions in the dark.
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: maximum of 14 days
- Proof of pregnancy: vaginal plug / sperm in vaginal smear] referred to as [day 0] of pregnancy
- Further matings after two unsuccessful attempts: not specified
- After successful mating each pregnant female was caged (how): Following evidence of mating (designated as day 0 post coitum) the males were returned to their original cages and females were transferred to individual cages.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The stability and homogeneity of the test item formulations were determined by Harlan Laboratories Ltd. (Shardlow, UK, Analytical Services). Results show the formulations to be stable for at least 21 days.

Samples of the test item formulation were taken and analyzed for concentration of Resin acids and rosin acids, maleated, esters with pentaerythritol, CAS NO. 94581-17-6 at Harlan Laboratories Ltd., Shardlow, UK, Analytical Services. The results indicate that the mean prepared dietary admixture concentrations were between 87% and 109% of the nominal concentration, confirming the accuracy of the mixing procedure.
Duration of treatment / exposure:
up to eight weeks (including a two week pre-pairing phase, pairing, gestation and early lactation for females)
Frequency of treatment:
Test item was administered continuously in the diet.
Doses / concentrationsopen allclose all
Dose / conc.:
0 ppm
Remarks:
Control
Dose / conc.:
3 000 ppm
Remarks:
Low Concentration (Mean Achieved Dosage Level equivalent to 179.3 mg/kg bw/day in males and 221.5 mg/kg bw/day in females)
Dose / conc.:
7 500 ppm
Remarks:
Intermediate Concentration (Mean Achieved Dosage Level equivalent to 441.9 mg/kg bw/day in males and 529.7 mg/kg bw/day in females)
Dose / conc.:
18 000 ppm
Remarks:
High Concentration (Mean Achieved Dosage Level equivalent to 783.4 mg/kg bw/day in males and 1508.1 mg/kg bw/day in females). Lower dietary concentration of 12000 ppm for both sexes from Day 22. Lower dietary concentration of 15000 ppm for females during gestation and lactation.
No. of animals per sex per dose:
12/sex/concentration
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: The dose levels were chosen in consultation with the Sponsor and were based on the results of previous toxicity work including a 14 Day range-finding study (Harlan Laboratories Limited Study Number: 41302583). The oral route was selected as the most appropriate route of exposure, based on the physical properties of the test item, and the results of the study are believed to be of value in evaluating the toxicological properties of the test item.

- Rationale for animal assignment (if not random): Animals were randomly allocated to treatment groups using a stratified body weight randomization procedure and the group mean body weights were then determined to ensure similarity between the treatment groups. The cage distribution within the holding rack was also randomized based on a Latin square arrangement to equalize environmental conditions or far as possible between the treatment groups. The animals were uniquely identified within the study by an ear punching system routinely used in these laboratories.

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All animals were examined for overt signs of toxicity, ill-health and behavioral change once daily.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were recorded on day 1 and then weekly for males until termination and weekly for females until pairing. During the pairing phase females were weighed daily until mating was confirmed. Body weights were then recorded for females on days 0, 7, 14 and 20 post coitum, and on days 1 and 4 post partum. Body weights were also recorded at terminal kill.

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; During the pre-pairing period, weekly food consumption was recorded for each cage of adults. This was continued for males after the mating phase. For females showing evidence of mating, food consumption was recorded for the periods covering post coitumdays 0-7, 7-14 and 14-20. For females with live litters, food consumption was recorded on days 1 and 4 post partum
- Food efficiency (the ratio of body weight change/dietary intake) was calculated retrospectively for males throughout the study period (with the exception of the mating phase) and for females during the pre-pairing phase. Due to offspring growth and milk production, food efficiency could not be accurately calculated for females during gestation and lactation.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: Water intake was measured daily during the pre-pairing phase of the study.

OTHER:

Functional Observations
Prior to the start of treatment and at weekly intervals thereafter, all animals were observed for signs of functional/behavioral toxicity. Functional performance tests were also performed on five selected males and females from each dose level, prior to termination, together with an assessment of sensory reactivity to various stimuli.

Laboratory Investigations
Haematological and blood chemical investigations were performed on five males and five females selected from each test and control group prior to termination (day 42 for males and day 4 post partum for females). Blood samples were obtained from the lateral tail vein. Where necessary repeat samples were taken by cardiac puncture at termination. Animals were not fasted prior to sampling

Pregnancy and Parturition
Each pregnant female was observed at least three times a day (early morning, mid-day and as late as possible during the normal working day) around the period of expected parturition. Observations were carried out at least twice (early morning and as late as possible during the normal working day) at weekends and public holidays. The following was recorded for each female:

i. Date of pairing
ii. Date of mating
iii. Date and time of observed start of parturition
iv. Date and time of observed completion of parturition

Litter Data
On completion of parturition (day 0 post partum), the number of live and dead offspring was recorded. Offspring were individually identified within each litter by tattoo on day 1 post partum. For each litter the following was recorded:

i. Number of offspring born
ii. Number of offspring alive recorded daily and reported on days 1 and 4 post partum
iii. Sex of offspring on days 1 and 4 post partum
iv. Clinical condition of offspring from birth to day 5 post partum
v. Individual offspring weights on days 1 and 4 post partum (litter weights were calculated retrospectively from this data)

All live offspring were assessed for surface righting reflex on day 1 post partum.
Oestrous cyclicity (parental animals):
Animals were paired on a 1 male: 1 female basis within each dose group, for a period of up to fourteen days. Males 76 and 78 from the high dose group were killed on day 7 of the pairing period due to poor clinical condition and were replaced with Males 73 and 75 respectively, from the same dose group, which had successfully mated. Cage tray-liners were checked each morning for the presence of ejected copulation plugs and each female was examined for the presence of a copulation plug in the vagina. A vaginal smear was prepared for each female and the stage of estrus or the presence of sperm was recorded. The presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating (day 0 of
gestation).
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: [yes/no]
- If yes, maximum of [...] pups/litter ([...]/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in [F1 / F2 / F3] offspring:
[number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), presence of nipples/areolae in male pups, other:]

GROSS EXAMINATION OF DEAD PUPS:
[no / yes, for external and internal abnormalities; possible cause of death was/was not determined for pups born or found dead.]

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY:

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY:
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals: Adult males were killed by intravenous overdose of pentobarbitane agent followed by exsanguination on day 43/44.
- Maternal animals: All surviving animals: Adult females were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on day 5 post partum. Female 23 from the control group was killed on day 4 post partumdue to the erroneous termination of the litter on this day (see Deviations from Study Plan).

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.
For all females, the uterus was examined for signs of implantation and the number of uterine implantations in each horn was recorded. This procedure was enhanced, if necessary, by staining the uteri with a 0.5% ammonium polysulphide solution (Salewski 1964). The corpora lutea were also counted. All adult animals and offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table 6. were prepared for microscopic examination and weighed, respectively.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at [#?] days of age.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

GROSS NECROPSY
- Gross necropsy consisted of [external and internal examinations including the cervical, thoracic, and abdominal viscera.]

HISTOPATHOLOGY / ORGAN WEIGTHS
The tissues indicated in Table [#] were prepared for microscopic examination and weighed, respectively.

Surviving offspring were terminated via intracardiac overdose
of a suitable barbiturate agent.
Statistics:
See ''Any other information on materials and methods incl. tables'' for information on statistics
Reproductive indices:
Mating Performance and Fertility
The following parameters were calculated from the individual data during the mating period of the parental generation:
i. Pre-coital Interval : Calculated as the time elapsing between initial pairing and the observation of positive evidence of mating.
ii. Fertility Indices: For each group the following were calculated:

Mating Index (%) = (Number of animals mated / Number of animals paired) x 100
Pregnancy Index (%) = (Number of pregnant females / Number of animals mated) x 100

Gestation and Parturition Data
The following parameters were calculated from individual data during the gestation and parturition period of the parental generation:
i. Gestation Length : Calculated as the number of days of gestation including the day for observation of mating and the start of parturition.
ii. Parturition Index: The following was calculated for each group:

Parturition Index (%) = (Number of females delivering live offspring / Number of pregnant females) x 100
Offspring viability indices:
The standard unit of assessment was considered to be the litter, therefore values were first calculated for each litter and the group mean was calculated using their individual litter values. Group mean values included all litters reared to termination (Day 5 of age).

i. Implantation Losses (%)
Group mean percentile pre-implantation and post-implantation loss were calculated for each female/litter as follows:

Pre–implantation loss (%) = ((Number of corpora lutea - Number of implantation sites) / (Number of corpora lutea)) x 100
Post–implantation loss (%) = ((Number of implantation sites - Number of offspring born) / (Number of implantation sites)) x 100

ii. Live Birth and Viability Indices
The following indices were calculated for each litter as follows:

Live Birth Index (%) = (Number of offspring alive on Day 1 / Number of offspring born) x 100
Viability Index (%) = (Number of offspring alive on Day 4 / Number of offspring alive on Day 1) x 100

iii. Sex Ratio (% males)
Sex ratio was calculated for each litter value on Days 1 and 4 post partum, using the following formula:

(Number of male offspring / Total number of offspring) x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Description (incidence and severity):
There were no clinical signs apparent for those animals that survived to scheduled necropsy at 3000, 7500 and 18000/12000 ppm.
Mortality:
mortality observed, treatment-related
Description (incidence):
There were three unscheduled and treatment-related deaths on the study, all occurring at the highest dietary concentrations.

Male number 76, receiving diet contraining 18000 ppm, was killed on day 22 of the study after showing marked body weight loss, piloerection, emaciation and dehydration. Necropsy revealed increased pelvic space in the left kidney, dark liver, reddened lungs and small seminal vesicles.

Male number 78, receiving diet containing 18000 ppm, was also killed on day 22 of the study after showing marked body weight loss, piloerection, emaciation and diuresis. Necropsy revealed reddened lungs and small seminal vesicles.

Female number 96 receiving diet containing 18000/12000 ppm showed hunched posture and piloerection on day 24 and 25 of the study. On day 25, these signs were also accompanied by dehydration and the animal was killed and subjected to macroscopic necropsy. This revealed an increased pelvic space in a pale left kidney, thickened stomach, and dark foci and sloughing of the glandular region of the stomach. While this death occurred when the animal was receiving diet containing 12000 ppm, it is considered that these signs reflected the previous exposure to diet containing 18000 ppm.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Males:
Males at the 18000 ppm dietary level, showed notable mean body weight loss during the first week of dietary exposure, with differences attaining statistical significance. While recovery of body weight gain was apparent during the following week, body weight gain still remained statistically significantly lower than control. During Week 3, a number of individual animals showed marked body weight loss, resulting in a notable mean body weight loss at this dosage, although differences were not statistically significant when compared to control. The extent of the individual body weight losses observed was sufficient to trigger a decision to reduce the dietary dosage for high dietary level animals. Following the reduction in dietary concentration, no further mean body weight losses or statistically significant differences in body weight gain were apparent although overall body weight gain at the end of the study for these animals remained notably lower than control.

Body weight gains for males receiving diet containing 7500 ppm were generally lower than control throughout the study and although differenceswere only statistically significant on one occasion (days 22-29), statistically significant lower overall body weight gain was also apparent at the end of the study for these animals.

Body weight gains for males receiving diet containing 3000 ppm were also generally lower than control throughout the study and lower overall body weight gain was also apparent at the end of the study, however differences from control showed no statistical significance. Animals at the age used on this study are no longer on a steep growth curve and the differences observed were considered most likely to represent normal biological variation and reflect particularly good gain for the control males.

Females:
Females at the 18000 ppm dietary level, showed statistically significant mean body weight loss during the first week of dietary exposure. Recovery of body weight gain was apparent during the following week, with body weight gain slightly superior to control, although differences did not attain statistical significance. The high dietary concentration for females was scheduled to be reduced to 15000 ppm during gestation and lactation but was further reduced due to concerns regarding the body weight performance for some individual animals. Body weight gain of females at the 18000/12000 ppm dietary level was lower than control throughout gestation, with differences being most noticeable during the last week of gestation. Body weight gain during the last week of gestation is increasingly influencedby the weight of the developing litter and the differences from control in body weight gain observed during this period was, at least in part, influenced by smaller litter size for these animals in comparison to their control counterparts. A marginal mean body weight loss during lactation was apparent for females at the 18000/ 12000 ppm dietary level, compared to a gain in the control group, despite the lower litter size for the treated animals.

For females receiving the 7500 ppm dietary level, body weight gains during the two week prepairing period were lower than control but differences failed to attain statistical significance. Body weight gains were also slightly lower than control throughout gestation and lactation, with statistically significant differences during the second week of gestation and also during lactation.

For females receiving the 3000 ppm dietary level, body weight gains during the two week prepairing period and during gestation and lactation were considered to be unaffected by dietary exposure to the test item.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Males:
Males at the 18000 ppm dietary level, showed notably lower mean food consumption compared with control, during the first week of dietary exposure. While some recovery of food intake was apparent during Week 2, food consumption remained noticeably lower than control. Following the pairing period, food consumption was still noticeably lower than control, despite the reduction in dietary concentration to 12000 ppm at this stage of the study.

For males at the 7500 ppm dietary level, food consumption was similar to control during the prepairing phase of the study. However, food intake was slightly lower than control during the post-pairing period.

Food consumption of males at the 3000 ppm dietary level was considered to have been unaffected by dietary exposure to the test item throughout the study.

Females:
Food consumption of females at all dietary levelswas considered to have been unaffected by dietary exposure during the pre-pairing phase of the study. Food consumption of females at the 18000/12000 ppm dietary level was statistically significantly lower than control throughout gestation and lactation. The differences were particularly noticeable during the late gestation and the lactation phases of the study and may have been influenced by reduced demand on the dams resulting from a lower litter size.

Food consumption of females at the 7500 ppm dietary level was lower than control throughout gestation, but differences were not marked and only attained statistical significance during the second week of gestation. However food intakeduring lactation was clearly lower than control with differences attaining statistical significance.

Food consumption of females at the 3000 ppm dietary level was considered to have been unaffected by dietary exposure during the gestation and lactation phases of the study.
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
At the 18000 ppm dietary level, food conversion efficiency was lower for both sexes during the first week of dietary exposure (a group mean body weight loss was observed for both sexes). There was no obvious effect on food utilisation of either sex during the remaining pre-pairing phase or for males during the post-pairing phase of the study.

At the 3000 and 7500 ppm dietary level, food conversion efficiency appeared unaffected by dietary exposure of either sex during the pre-pairing phase or for males during the post-pairing phase of the study.
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
Males at the 18000 ppm dietary level, showed lower mean water consumption compared with control, throughout the two week pre-pairing period. This may be related to the lower food consumption observed for these animals during this period.

For males at the 7500 ppm dietary level, water intake during the pre-pairing period appeared lower than control from day 11 of the study. Prior to this, values did not indicate any consistent effect of dietary exposure to the test item.

For males at the 3000 ppm and females at all dietary levels, water consumption during the prepairing period was considered to have been unaffected by dietary exposure to the test item.
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
At the 18000/12000 ppm exposure level, males showed statistically significant lower mean corpuscular haemoglobin and females statistically significant lower mean cell haemoglobin concentration compared with control; however differences for these treated animals were all within the historical control range.

Females at all dietary levels, had statistically significant lower neutrophil counts compared with the control group, with means of 2.500, 1.104*, 1.258** and 0.0988* for control, low, intermediate and high dietary groups respectively. Mean values showed no consistent relationship to dietary exposure levels and among the test-item exposed animals only one individual value at 3000 ppm was below the historical control range (0.45-2.84) while two control values exceeded this historical range. It is considered that the observed differences reflect high control values rather than any effect of treatment.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
For females at the 7500 and 18000/12000 ppm dietary levels, total protein, albumin levels and albumin/globulin ratio were lower than control with differences attaining statistical significance. Mean values for both protein and albumin showed a consistent relationship to dietary exposure. The majority of individual values for these parameters were within the respective historical control ranges; one albumin value at the 18000/12000 ppm dietary level and one albumin/globulin ratio value at the 7500 ppm dietary level were outside their respective ranges and one control albumin value exceeded the historical range. Statistically significantly lower total protein and albumin levels were also observed for males at the 18000/ 12000 ppm dietary level but there was no accompanying statistically significantly effect on albumin/globulin ratio; only one individual value for total protein was below the historical control range for these treated animals. For males at all dosages, higher levels of potassium attained statistical significance compared to control; only two individual values at both the 3000 and 7500 ppm dietary level and one value at the 18000/ 12000 ppm dietary level exceeded the historical control range.

For both sexes at all dietary concentrations, higherlevels of total billirubin attained statistical significance when compared with control, with mean values showing a consistent dosage relationship; the majority of individual values at the 7500 and 18000/12000 ppm dietary levels exceeded the historical control range.

For females at the 7500 ppm dietary level and both sexes at the 18000/12000 ppm dietary level, alkaline phosphatase was lower than control with differences attaining statistical significance; the majority of individual values for these treated females were outside the historical control range but all values for these treated males were within the historical control range.

For females at the 18000/12000 ppm dietary level, lower inorganic phosphorus level attained statistical significance compared to control. However, as all individual values for these treated animals were within the historical control range and two control values exceeded this historical range, this finding was considered unlikely to be related to treatment and of no toxicological significance.

For females at the 7500 ppm dietary level, lower sodium levels also attained statistical significance compared to control; however only one individual value for these treated animals was outside the historical control range. In the absence of any similar findings at 18000/12000 ppm, this finding was considered to be incidental and unlikely to be related to treatment.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Behavioral Assessments
There were no treatment-related changes in the behavioural parameters at dietary concentrations of 3000, 7500 or 18000/12000 ppm.

One female at 3000 ppm dietary level showed palprebral closure on day 4 post partumbut this isolated finding, in the absence of any effects at higher dietary levels, was considered to be incidental and unrelated to treatment.

Functional Performance Tests
There were no changes in functional performance considered to be related to dietary exposure at 3000, 7500 or 18000/12000 ppm.

Sensory Reactivity Assessments
There were no inter-group differences in sensory reactivity scores that were considered to be related to dietary exposure at 3000, 7500 or 18000/12000 ppm.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
The test-item-related effects occurred with a greater incidence and/or severity than in controls or were present in treated animals but not in controls. They can be summarized as follows:

Adrenal Cortex:
- Minimal or mild diffuse hypertrophy of the zona glomerulosa in both sexes given diets containing 7500 or 18000/12000 ppm.
- Minimal diffuse atrophy of the zona fasciculata observed in 3/12 males given diets containing 7500 ppm and most animals of either sex given diets containing 18000/ 12000 ppm.

Bone Marrow:
- Minimal or mild increase in adipose tissue in one or more of the bone marrow samples examined from each animal in both sexes given diets containing 18000/12000 ppm and males given diets containing 7500 ppm.

Kidneys:
- Mild or moderate foci of basophilic tubules in 2/12 males and 1/12 females given diets containing 18000/12000 ppm.
- Minimal or mild single cell death in the affected tubules of these animals.
- Minimal to moderate pelvic dilatation, unilateral or bilateral, in both sexes given diets containing 18000/12000 and males given diets containing 7500 ppm.
- Mild hypertrophy of the collecting ducts in the papilla was seen in 2/12 males and 3/12 females given diets containing 18000/12000 ppm.

Liver:
- Minimal diffuse atrophy of hepatocytes in all the animals given diets containing 18000/12000 ppm and in 7/12 males and 9/12 females given diets containing 7500 ppm.

Mesenteric Lymph Node:
- Minimal to moderate intrasinusal congestion/erythrophagocytosis in both sexes given diets containing 18000/12000 ppm and males given diets containing 7500 ppm.

Pituitary Gland:
- Minimal diffuse atrophy of pituicytes in 7/11 females given diets containing 7500 and 10/12 females given diets containing 18000/12000 ppm.

Secondary Sex Glands of Males:
- Minimal or mild reduction in secretion was observed in the seminal vesicles of 5/12 males given diets containing 18000/12000 ppm one of which also had minimal reduced secretion in the prostate gland.

Spleen:
- Extramedullary haematopoiesis was lower in females only given diets containing 7500 or 18000/12000 ppm than in controls.

Thymus:
- Minimal or mild decreased cortical lymphocytes in both sexes given diets containing 18000/12000 ppm and females given diets containing 7500 ppm.

Urinary Bladder:
- Minimal to moderate diffuse hyperplasia of the urothelium in females given diets containing 7500 ppm and both sexes given diets containing 18000/12000 ppm.
- Associated minimal to moderate chronic active inflammation in most females given diets containing 7500 ppm and most females and some males given diets containing 18000/ 20000 ppm.
- In a few affected animals of the highest dietary exposure group erosion or ulceration of the mucosa.
- In a female from the highest dietary exposure group, minimal focal submucosal haemorrhage.
- Minimal inflammatory cell infiltration of the submucosal in 2/7 females given diets containing 7500 ppm.

The test item-related changes in urinary bladder and kidneys were considered adverse and due to an irritant effect of the compound or most likely, a metabolite to the urothelium. More severe effects occurring in the urinary bladder probably because of the longer time that the mucosa is exposed to the irritant.

With the exception of the change seen in the mesenteric lymph node the test-item-related effects in the other tissues could all be the result ofcomparative differences in food consumption and body weight gain and, in the zona glomerulosa, the effects of dehydration on electrolyte imbalance, Levin et al(1993).

The change seen in the mesenteric lymph nodes was unexplained but was considered of minor biological importance and therefore non-adverse.

All of the other histopathological findings encountered were considered to have occurred spontaneously or post mortem. In addition to the routine evaluation of the testes, the seminiferous tubules of the testes were evaluated with respect to their stage in the spermatogenic cycle and the integrity of the various cell types present within the different stages. No treatment related abnormalities were noted.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Fertility: Fertility as assessed by pregnancy rate was unaffected by dietary exposure at all dietary levels.

Gestation Length: Gestation lengths (where mating was observed) were between 22 and 23½ days and the distribution of gestation lengths for treated females was essentially similar to control and did not indicate any effect of dietary exposure.

At the 18000/12000 ppm dietary level, mean corpora lutea number was lower than control, with differences attaining statistical significance. Although subsequent pre and post implantation losses were similar to control, mean implantation count and litter size at birth/post-natal day 1 remained statistically significantly lower than control. One female (No. 85) showed total litter loss post partum, however post natal survival for the remaining litters was similar to control, although litter size continued to be significantly lower than control to post-natal day 4 of age. Sex ratio was unaffected indicating that maternal dietary exposure produced no selective effect on the survival of the offspring at this dietary level.

There was considered to be no obvious effect ofdietary exposure on the numbers of corpora lutea, implantations, pre and post implantation losses, litter size at birth/post-natal day 1 and post natal day 4 of age and post natal survival at the 3000 or 7500 ppm dietary levels. At 3000 ppm, lower corpora lutea count attained statistical significance when compared to control but this was considered not to represent an effect of treatment, as no statistically significant effect was apparent for corpora lutea count at 7500 ppm.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
In addition to the routine evaluation of the testes, the seminiferous tubules of the testes were evaluated with respect to their stage in the spermatogenic cycle and the integrity of the various cell types present within the different stages. No treatment related abnormalities were noted.
Reproductive performance:
effects observed, non-treatment-related
Description (incidence and severity):
Mating:
There was no obvious effect of dietary exposure on mating performance with the majority of matings occurring within the first four days of pairing (i.e. at the first estrus opportunity).

As previously discussed in the mortality section, female 96 (receiving 18000/12000 ppm) was killed in extremisduring the pairing period (study day 25). This animal did not show any evidence of mating prior to being terminated, however this was considered not to indicate any effect on mating performance at this dietary level.

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
ca. 3 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Systemic Toxicity
Remarks on result:
other: Mean Achieved Dosage Level equivalent to 179.3 mg/kg bw/day in males and 221.5 mg/kg bw/day in females
Key result
Dose descriptor:
NOAEL
Effect level:
ca. 7 500 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Reproductive Toxicity
Remarks on result:
other: Mean Achieved Dosage Level equivalent to 441.9 mg/kg bw/day in males and 529.7 mg/kg bw/day in females

Target system / organ toxicity (P0)

Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
7 500 ppm
System:
urinary
Organ:
bladder
kidney
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
The clinical signs apparent for offspring on the study were typical for the age observed and did not indicate any clear effect of maternal dietary exposure on offspring development at 3000, 7500 and 18000/12000 ppm.

At 7500 and 18000/12000 ppm there was a slightly higher incidence of clinical signs for the offspring however, these tended to be concentrated in only a few litters, and overall did not indicate a clear effect of maternal dietary exposure on offspring development. These findings included no milk visable in stomach (2 and 3 litters at 7500 ppm and 18000/12000 ppm respectively), cold (1 litter at 18000/12000 ppm), weak (1 litter at 18000/12000 ppm), small (2 and 3 litters at 7500 ppm and 18000/12000 ppm respectively) and distended abdomen (1 litter at 7500 ppm).
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
In total 12, 12, 12 and 10 females from the control, 3000, 7500 and 18000/12000 ppm dietary groups gave birth to a live litter and successfully reared young to post-natal day 4. Additionally, one female at the 18000/12000 ppm dietary level showed total litter loss post partum.

At the 18000/12000 ppm dietary level one female (No. 85) showed total litter loss post partum, however post natal survival for the remaining litters was similar to control, although litter size continued to be significantly lower than control to post-natal day 4 of age. There was considered to be no obvious effect of dietary exposure on litter size at birth/post-natal day 1 and post natal day 4 of age and post natal survival at the 3000 or 7500 ppm dietary levels.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At the 18000/12000 ppm dietary level, offspring body weight on days 1 and 4 and subsequent body weight gain to day 4 was lower than concurrentcontrol, despite the lower litter size at this dietary level, with differences attaining statistical significance. Litter weights were also statistically significantly lower than control,reflecting both lower pup body weights and the smaller litter size.

At the 7500 ppm dietary level, offspring body weight on post-natal day 1 and 4 post partumand subsequent body weight gain to post-natal day 4 were also lower than concurrent control, with differences attaining statistical significance. Lower litter weight also attained statistical significance on post-natal day 4 of age.

At the 3000 ppm dietary level, pup body weight on post-natal day 1 and subsequent pup body weight gain to post-natal day 4 of age and litter weights at post-natal day 1 and 4 were considered to be unaffected by maternal dietary exposure.
Sexual maturation:
no effects observed
Description (incidence and severity):
Sex ratio was unaffected indicating that maternal dietary exposure produced no selective effect on the survival of the offspring at all dietary levels.

Developmental neurotoxicity (F1)

Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
At the 7500 and 18000/12000 ppm dietary level, offspring performance during assessment of surface righting was inferior to control, although the mean values did not show a relationship to dietary exposure level. Additionally, in the high exposure group, all of the offspring from the one litter that died before scheduled termination on day 5 failed to showed righting reflex.

At the 3000 ppm dietary level, surface righting ability of the offspring appeared to be unaffected by maternal dietary exposure to the test item.

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
ca. 3 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Survival, growth and development
Remarks on result:
other: Mean Achieved Dosage Level equivalent to 179.3 mg/kg bw/day in males and 221.5 mg/kg bw/day in females

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
yes
Lowest effective dose / conc.:
7 500 ppm
Treatment related:
yes
Relation to other toxic effects:
not specified
Dose response relationship:
yes
Relevant for humans:
not specified

Any other information on results incl. tables

Table 7. Group Mean Body Weight Values - Females (P0)

 

Group

 

Day Numbers

 

Gestation

Lactation

 

0

7

14

20

1

4

Control

Mean

226.2

255.8

284.3

347.2

262.8

277.1

S.D.

9.3

9.6

11.5

21.0

10.8

8.6

N

12

12

12

12

12

12

 

Low Concentration

Mean

227.3

253.5

281.9

341.5

261.2

269.7

S.D.

7.8

9.5

12.5

18.6

11.8

10.7

N

11

11

11

11

12

12

 

Intermediate

Concentration

Mean

217.0*

244.3*

266.6**

319.9**

244.0**

244.6***

S.D.

9.6

12.4

13,0

21.0

13.4

13.9

N

12

12

12

12

12

12

 

High Concentration

Mean

206.8***

227.4***

245.2***

275.0***

219.8***

218.8***

S.D.

9.0

9.1

12.0

9.6

10.1

10.7

N

10

10

10

10

10

10

Table 8. Group Mean Body Weight Gains – P0 - Males (Increase in Body Weight (g))

 

 

Day Numbers Relative to Start Date

 

 

 

 

 

 

 

 

Abs Gain

% Gain

Group

From

1

8

15

22

29

36

1

1

To

8

15

22

29

36

43

43

43

 

Control

Mean

27.2

21.3

7.8

18.1

16.8

7.2

98.3

30.2

S.D.

6.8

5.5

5.0

7.3

7.2

6.9

25.1

7.2

N

12

12

12

12

12

12

12

12

 

Low Concentration

Mean

21.6

18.2

8.1

11.6

13.0

4.9

77.3

24.1

S.D.

9.5

7.8

7.5

5.1

3.9

6.7

25.2

8.0

N

12

12

12

12

12

12

12

12

 

Intermediate

Concentration

Mean

21.8

17.0

5.3

10.1*

14.8

5.5

74.3*

23.1

S.D.

6.4

6.1

6.8

6.2

6.5

4.9

25.8

7.7

N

12

12

12

12

12

12

12

12

 

High Concentration

Mean

-6.9**

12.3**

-12.6

21.4

15.3

4.9

47.9***

15.0

S.D.

9.9

5.4

29.5

11.3

4.3

3.4

14.8

4.4

N

12

12

12

10

10

10

10

10

Table 9. Group Mean Body Weight Gains – P0 - Females

 

 

 

Increase in Body Weight (g)

Cumulative Body Weight Change (g)

Days

 

Group

 

Gestation

Lactation

Gestation

From

0

7

14

1

0

0

To

7

14

20

4

14

20

 

Control

Mean

29.7

28.4

62.9

14.3

58.1

121.0

S.D.

5.2

7.2

16.0

6.8

9.7

19.6

N

12

12

12

12

12

12

 

Low Concentration

Mean

26.3

28.4

59.6

8.5

54.6

114.3

S.D.

5.3

4.6

7.5

4.9

8.1

14.0

N

11

11

11

12

11

11

 

Intermediate

Concentration

Mean

27.3

22.3*

53.3

0.6***

49.6

102.9*

S.D.

7.3

6.0

9.2

8.5

9.7

16.7

N

12

12

12

12

12

12

 

High Concentration

Mean

20.6**

17.8***

29.8***

-1.0***

38.4***

68.2***

S.D.

6.8

4.4

7.5

5.8

9.1

11.5

N

10

10

10

10

10

10

Table 10. Group Mean Food Consumptions – P0 - Females

 

 

Group

 

Day Numbers

 

Gestation

Lactation

From

0

7

14

1

To

7

14

20

4

Control

Mean

19.5

22.5

22.7

28.4

S.D.

1.3

2.1

1.5

3.8

N

12

12

12

12

Low Concentration

Mean

19.3

21.7

23.4

27.0

S.D.

1.5

2.0

2.0

6.1

N

11

11

11

12

Intermediate

Concentration

Mean

18.1

19.3**

21.7

19.9***

S.D.

2.1

2.1

1.6

2.8

N

12

12

12

12

High Concentration

Mean

17.0*

19.0**

17.7***

14.5***

S.D.

1.3

2.8

1.4

3.6

N

10

10

10

10

Table 11. Group Mean Litter Size and Litter Weights

 

 

 

 

Group

 

Number

of

Corpora

Lutea

Number of

Implantation

Sites

Total

Number of

Offspring

Born

Number of Live

Offspring

Litter Weight

(g)

Offspring Weight (g)

Offspring

Body Weight Change

(g)

 

 

 

 

 

Day 1

Day 4

Day 1

Day 4

Day 1

Day 4

Day 1

Day 4

Days 1-4

 

 

 

 

 

 

 

 

Males

females

Males

females

Males

females

 

Control

Mean

14.3

12.9

12.0

11.8

11.8

71.80

102.87

6.24

6.09

9.08

8.92

2.84

2.83

S.D.

2.1

3.6

3.3

3.3

3.2

18.5

24.39

0.55

0.55

1.16

1.15

0.66

0.61

N

12

12

12

12

12

12

12

12

12

12

12

12

12

 

Low Concentration

Mean

11.8*

11.8

11.2

11.2

11.0

67.43

96.67

6.33

5.95

9.12

8.80

2.79

2.84

S.D.

2.8

2.7

2.7

2.7

2.5

13.76

17.91

0.69

0.63

1.07

1.05

0.47

0.48

N

12

12

12

12

12

12

12

12

12

12

12

12

12

 

Intermediate

Concentration

Mean

12.4

12.0

11.2

11.2

10.8

60.95

77.34**

5.63*

5.37**

7.45**

7.06**

1.82***

1.70***

S.D.

2.4

2.3

2.5

2.5

2.2

12.33

15.75

0.54

0.44

1.06

1.01

0.64

0.61

N

12

12

12

12

12

12

12

12

12

12

12

12

12

 

High Concentration

Mean

9.0**

8.3***

7.6**

7.6**

7.4**

39.43***

49.29***

5.39**

5.16***

6.92***

6.68**

1.53***

1.52***

S.D.

2.5

1.7

1.4

1.4

1.5

6.61

8.21

0.65

0.60

1.18

1.08

0.63

0.65

N

10

10

10

10

10

10

10

10

10

10

10

10

10

Table 12. Group Mean Blood Chemical Values – P0 - Males

Group

 

Total Prot.(g/dL)

Albumin

(g/dL)

K+

(mmol/L)

AP

(IU/L)

Bili

(mg/dL)

 

Control

Mean

6.832

3.92

4.530

142.8

0.096

S.D.

0.197

0.08

0.179

17.6

0.005

N

5

5

5

5

5

 

Low Concentration

Mean

6.794

3.78

4.966*

141.6

0.114*

S.D.

0.226

0.18

0.581

27.4

0.005

N

5

5

5

5

5

 

Intermediate

Concentration

Mean

6.684

3.74

4.916*

149.8

0.192**

S.D.

0.208

0.21

0.184

56.6

0.026

N

5

5

5

5

5

 

High Concentration

Mean

6.378*

3.58**

5.150*

95.4**

0.332**

S.D.

0.502

0.16

1.068

14.2

0.045

N

5

5

5

5

5

Table 13. Group Mean Blood Chemical Values – P0 - Females

Group

 

Total Prot.

(g/dL)

Albumin

(g/dL)

A/G Ratio

Na+

(mmol/L)

P

(mmol/L)

AP

(IU/L)

Bili

(mg/dL)

 

Control

Mean

6.874

3.96

1.366

147.0

1.52

136.8

0.058

S.D.

0.391

0.27

0.051

1.4

0.55

33.1

0.011

N

5

5

5

5

5

5

5

 

Low Concentration

Mean

6.600

3.76

1.328

148.2

1.38

167.4

0.082*

S.D.

0.420

0.34

0.105

4.1

0.60

62.5

0.022

N

5

5

5

5

5

5

5

 

Intermediate

Concentration

Mean

6.272**

3.44**

1.218*

141.0*

1.02

71.0*

0.188**

S.D.

0.200

0.15

0.102

4.8

0.41

19.2

0.085

N

5

5

5

5

5

5

5

 

High Concentration

Mean

5.934**

3.36**

1.284*

143.6

0.82*

47.6**

0.396**

S.D.

0.311

0.18

0.044

2.3

0.26

35.4

0.247

N

5

5

5

5

5

5

5

Table 14. Group Mean Organ Weights with Corresponding Relative (% of Body Weight) Organ Weights

 

Organ

 

Males

Females

0

Control

3000

ppm

7500

ppm

18000 /

12000

ppm

0

Control

3000

ppm

7500

ppm

18000 /

12000

ppm

 

Liver

Mean (g)

13.6882

13.2353

13.2295

12.9473**

11.7482

12.3408**

9.96312

8.85520

S.D.

1.51014

1.30389

1.75721

1.46364

1.20970

1.38803

0.90712

0.59784

N

12

12

12

10

12

12

12

10

 

Mean (%)

3.254

3.312

3.327

3.525**

4.164

4.516**

4.023

4.007

S.D.

0.159

0.201

0.263

0.259

0.314

0.433

0.236

0.289

N

12

12

12

10

12

12

12

10

 

Ovaries

Mean (g)

 

 

 

 

0.12826

0.10733**

0.09420*

0.08598

S.D.

 

 

 

 

0.01814

0.01303

0.01810

0.01552

N

 

 

 

 

12

12

12

10

 

Mean (%)

 

 

 

 

0.046

0.039**

 

0.038*

0.039

S.D.

 

 

 

 

0.007

0.005

0.006

0.007

N

 

 

 

 

12

12

12

10

 

Pituitary

Mean (g)

0.01471

0.01258*

0.01234*

0.01077*

0.01759

0.01716

0.01464*

0.01310**

S.D.

0.00286

0.00309

0.00156

0.00145

0.00271

0.00178

0.00250

0.00116

N

12

12

12

10

12

12

12

10

 

Mean (%)

0.004

0.003*

0.003*

0.003**

0.006

0.006

0.006**

0.006**

S.D.

0.001

0.001

0.000

0.000

0.001

0.001

0.001

0.000

N

12

12

12

10

12

12

12

10

 

Seminal Vesicles

Mean (g)

2.26419

2.05758

1.89807**

1.56437**

 

 

 

 

S.D.

0.33826

0.40448

0.27070

0.30683

 

 

 

 

N

12

12

12

10

 

 

 

 

 

Mean (%)

0.543

0.516

0.479**

0.429**

 

 

 

 

S.D.

0.095

0.099

0.061

0.091

 

 

 

 

N

12

12

12

10

 

 

 

 

 

Spleen

Mean (g)

0.76190

0.78678

0.64450

0.80042

0.67432

0.59530**

0.59402*

0.46908**

S.D.

0.10345

0.04341

0.11716

0.08103

0.05867

0.05576

0.05440

0.02662

N

5

5

5

5

5

5

5

5

 

Mean (%)

0.178

0.190

0.167

0.214

0.238

0.216**

0.233*

0.209**

S.D.

0.020

0.016

0.026

0.025

0.028

0.026

0.020

0.015

N

5

5

5

5

5

5

5

5

 

Uterus and Cervix

Mean (g)

 

 

 

 

0.70715

0.65747

0.68088

0.55625**

S.D.

 

 

 

 

0.09892

0.12481

0.15165

0.10713

N

 

 

 

 

12

12

12

10

 

Mean (%)

 

 

 

 

0.251

0.240

0.274

0.251**

S.D.

 

 

 

 

0.038

0.039

0.055

0.045

N

 

 

 

 

12

12

12

10

Applicant's summary and conclusion

Conclusions:
Based on the results this study the No Observed Adverse Effect Level (NOAEL) for systemic toxicity was considered to be 3000 ppm, principally due to effects on body weight gain, food consumption and adverse histopathological changes in the kidney and urinary bladder at 7500 ppm and 18000/12000 ppm.

The No Observed Adverse Effect Level (NOAEL)for reproduction was considered to be 7500 ppm due to lower corpora lutea count at 18000/12000 ppm.

The No Observed Adverse Effect Level (NOAEL) for survival, growth and development of the offspring was considered to be 3000 ppm due to effects on body weight, body weight gain and surface righting ability at 7500 ppm and 18000/12000 ppm.
Executive summary:

In a key combined repeat dose toxicity study with reproduction/developmental toxicity screening test, the test material (Resin acids and rosin acids, maleated, esters with pentaerythritol, CAS# 94581-17-6) was administered in the diet to three groups, each composed of twelve male and twelve female Wistar Han™:RccHan™:WIST strain rats, for up to eight weeks (including a two week pre-pairing phase, pairing, gestation and early lactation for females), at dietary concentrations of 3000, 7500 and 18000 ppm. The dietary concentration given to the high dietary concentration females during gestation and lactation was initially decreased to 15000 ppm to lessen the expected increase in achieved intake during these phases. However, due to adverse toxicity, the high dietary concentration was reduced to 12000 ppm for both sexes on study day 22. Estimated achieved dosages for males in Groups 2 to 4 during the study was 179.3, 441.9 and 783.4 mg/kg bw/day respectively. For females, it was 221.5, 529.7 and 1508.1 mg/kg bw/day during the pre-pairing phase, 237.0, 570.8 and 911.6 during gestation and 305.1, 610.9 and 794.0 mg/kg bw/day during lactation respectively. A control group of twelve males and twelve females were treated with basal laboratory diet.

 

Clinical signs, behavioural assessments, body weight change and food and water consumption were monitored during the study. Pairing of animals within each dose group was undertaken on a one male: one female basis within each treatment group on study day 15, with females subsequently being allowed to litter and rear their offspring to lactation day 5. During the lactation phase, daily clinical observations were performed on all surviving offspring, together with litter size and offspring weights and assessment of surface righting reflex. Extensive functional observations were performed on five males from each dose group after the completion of the pairing phase, and for five parental females from each dose group on day 4 post-partum.Haematology and blood chemistry were evaluated prior to termination on five males and females from each dose group. Surviving adult males were terminated on study day 43/44, followed by the termination of all surviving females and offspring on day 5 post-partum. All adult animals, including decedents were subjected to a gross necropsy examination and histopathological evaluation of selected tissues was performed.

 

There were three unscheduled and treatment-related deaths at the highest dietary level (18000 ppm). No signs of treatment-related clinical toxicity were apparent in any of the surviving animals at 3000, 7500 and 18000/ 12000 ppm. Behavioural assessments, functional performance, sensory reactivity assessments, and haematology parameters were unaffected by dietary exposure at 3000, 7500 and 18000/ 12000 ppm. Gross necropsy did not reveal any remarkable findings in rats of either sex.

 

At the 18000 ppm dietary concentration, notable mean body weight loss during the first week of dietary exposure and statistically significant lower body weight gain during Week 2, compared with control, was observed in males. During Week 3, a number of individual animals showed marked body weight loss, resulting in a mean bodyweight loss and the dietary concentration was lowered to 12000 ppm for the high dietary level group. No further mean body weight losses or statistically significant differences in body weight gain were apparent for the remainder of the study although the overall body weight gain at termination of the study remained statistically significantly lower than control. Females in the 18000 ppm group, showed notable mean body weight loss during the first week of dietary exposure but recovery of body weight gain was apparent during the following week. Body weight gain of females at 18000/12000 ppm was lower than control throughout gestation, particularly during the last week of gestation. During lactation, marginal mean body weight loss was apparent for females in the 18000/12000 ppm group compared with control. Body weight gains for males receiving diets containing 7500 ppm were generally lower than control throughout the study and statistically significant lower overall body weight gain was apparent for these animals at the end of the study. Body weight gains for females that received diets containing 7500 ppm were lower than control during the two week pre-pairing period and throughout gestation and lactation, although differences only attained statistical significance during the second week of gestation and during lactation. There was no obvious effect of dietary exposure to 3000 ppm on body weight gains for both sexes throughout the study.

 

At the 18000 ppm dietary concentration, males showed notably lower mean food consumption during the first and, to a lesser extent, second week of dietary exposure compared with control. Following the pairing period, food consumption remained lower than control, despite the animals only receiving 12000 ppm at this stage of the study. For females at the 18000/12000 ppm dietary concentration, food consumption was lower than control throughout gestation and lactation phases; these differences were particularly noticeable during late gestation and during lactation. At the 7500 ppm dietary concentration, food consumption for males was slightly lower than control during the post-pairing period. Food consumption for females was lower than control throughout gestation, and, to a greater extent, lactation. At the 3000 ppm dietary concentration, food consumption for each sex was considered to have been unaffected by dietary exposure to the test material.

 

At the 18000 ppm dietary concentration, food conversion efficiency was lower for both sexes during the first week of dietary exposure to the test item. At the 3000 and 7500 ppm dietary concentrations, food conversion efficiency for both sexes appeared to be unaffected by dietary exposure to the test material.

 

At the 18000 ppm dietary concentration, males showed lower mean water consumption compared with control, throughout the two week pre-pairing period. For males at the 7500 ppm dietary concentration, water intake during the pre-pairing period appeared lower than control from day 11 of the study; prior to this, values did not indicate any consistent effect of dietary exposure. For males at the 3000 ppm dietary concentration and females at all dietary levels, water consumption during the pre-pairing period was unaffected by dietary exposure.

 

For both sexes at all dietary concentrations, statistically significant higher levels of total bilirubin were observed when compared with control, with mean values showing a consistent relationship to test material exposure. The majority of individual values at the 7500 and 18000/12000 ppm dietary concentrations exceeded the historical control range. All individual values for the 3000 ppm dietary group were within the historical control range. For females at the 7500 and 18000/12000 ppm dietary concentrations, statistically significant lower total protein, albumin and albumin/globulin ratio were observed compared with control; however the majority of individual values were within the historical control range. Statistically significant lower total protein and albumin levels were also observed for males at the 18000/12000 ppm dietary concentrations, however, the majority of individual values were within the historical control range. For females at the 7500 ppm dietary level and both sexes at the 18000/12000 ppm dietary level, alkaline phosphatase was lower than control with differences attaining statistical significance; the majority of individual values for these exposed females were below the historical control range but all values for these exposed males were within the historical control range. For males at all dietary concentrations, statistically significant higher levels of potassium were observed compared to control, however, the majority of individual values were within the historical control range.

 

Although macroscopic necropsy findings at termination did not indicate any obvious effect of dietary exposure to the test material at 3000, 7500 and 18000/12000 ppm, a number of organ weights attained statistical significance compared to control: Males at the 18000/12000 ppm dietary level showed lower absolute liver weight and higher relative liver weights compared to control, although the majority of individual values were within the historical control range. Microscopic evaluation revealed minimal diffuse atrophy of hepatocytes for both sexes at 18000/12000 ppm and, to a lesser extent, at 7500 ppm.

 

Females at the 7500 and 18000/12000 ppm dietary levels, showed lower absolute and relative pituitary weight compared to control, although the majority of individual values were within the historical control range. Microscopic evaluation revealed minimal diffuse atrophy of pituicytes for females at the 18000/12000 ppm and, to a lesser extent, at the 7500 ppm dietary level. Males receiving diets containing 7500 ppm or 18000/12000 ppm of the test item showed lower absolute and relative seminal vesicles weights compared to control. At the 18000/12000 ppm exposure level a number of individual seminal vesicle values were outside the historical control range and microscopic evaluation revealed a minimal or mild reduction in secretion in the seminal vesicles. This finding at the high dietary exposure level was therefore considered to be treatment related. For males receiving 7500 ppm of the test material all individual seminal vesicles weights were within the historical control and in the absence of any supporting histopathological change, the decrease in seminal vesicle weight at this dietary concentration was considered not to be of toxicological significance. Females at all dietary levels showed lower absolute and body weight relative spleen weights compared with control but all individual absolute and body weight relative values for treated animals were within the historical control range. Microscopic evaluation revealed lower extramedullary haematopoiesis for females at the 7500 or 18000/12000 ppm dietary levels compared to control, therefore the lower spleen weights at these exposure levels were considered to be related to treatment. At the 3000 ppm exposure level, there was no evidence of histopathological change and the lower spleen weights were considered to be of no toxicological significance.

 

These differences in organ weight and accompanying microscopic changes were considered to reflect comparative differences in food consumption and body weight gain and, in the zona glomerulosa, the effects of dehydration on electrolyte imbalance, (Levin et al.,1993). Other microscopic changes also considered to be attributable to these differences included: Minimal or mild diffuse hypertrophy of the zona glomerulosa of the adrenal cortex in both sexes at the 7500 or 18000/12000 ppm dietary levels and minimal diffuse atrophy of the zona fasciculata of the adrenal cortex in both sexes at the 18000/12000 ppm dietary level and, to a lesser extent, males at the 7500 ppm dietary level.

 

Minimal or mild increase in adipose tissue was observed in the bone marrow samples of males at the 7500 ppm dietary level and in both sexes at the 18000/12000 ppm dietary level. Minimal reduced secretion in the prostate gland of one male was observed at the 18000/12000 ppm dietary level. Minimal or mild decreased cortical lymphocytes of the thymus was observed in females at the 7500 ppm dietary level and in both sexes at 18000/12000 ppm dietary level.

 

Other microscopic changes, considered to be more serious in nature, were also observed. For the kidney, mild or moderate foci of basophilic tubules were apparent for a few animals at the 18000/12000 ppm dietary level, these animals also showed minimal or mild single cell death in the affected tubules. Minimal to moderate pelvic dilatation was also apparent for males at the 7500 ppm dietary level and both sexes at the 18000/12000 ppm dietary level and mild hypertrophy of the collecting ducts in the papilla was seen a few animals at the 18000/ 12000 ppm dietary level. For the urinary bladder, minimal to moderate diffuse hyperplasia of the urothelium, with associated minimal to moderate chronic active inflammation, was apparent for females at the 7500 ppm dietary level and in both sexes at the 18000/12000 ppm dietary level. At the highest exposure level, a few affected animals also showed erosion or ulceration of the mucosa and one female showed minimal focal submucosal haemorrhage. Two females at the 7500 ppm dietary level also showed minimal inflammatory cell infiltration of the submucosa. The observed changes in the urinary bladder and kidneys were considered adverse and due to an irritant effect of the test item, or a metabolite, to the urothelium. The more severe effects apparent in the urinary bladder was probably due to the longer time that the urinary bladder mucosa was exposed to the test item or metabolite. Microscopic changes were also apparent in the mesenteric lymph nodes, with minimal to moderate intrasinusal congestion/erythrophagocytosis being observed in males at the 7500 ppm dietary level and in both sexes at the 18000/12000 ppm dietary level.

 

Mating performance, pregnancy rate, and gestation length were unaffected by dietary exposure to the test material. At the 18000/12000 ppm dietary concentration, the mean number of corpora lutea was lower than control, resulting in a lower number of implantations and litter size on post-natal days 1 and 4. However, no obvious effect on pre-natal and post-natal survival or sex ratio was observed at this exposure level. At the 3000 and 7500 ppm dietary concentrations, there were no obvious effects on numbers of corpora lutea, implantations, pre-and post implantation losses, litter size or sex ratio at birth/postnatal day 1 and post-natal day 4 of age or on post-natal pup survival.

 

At the 18000/12000 ppm dietary concentration, offspring bodyweight weight and litter weights on post-natal days 1 and 4 and offspring body weight gain to post-natal day 4 were lower than control. Offspring performance during assessment of surface righting on post-natal day 1 was also inferior to control at the highest exposure level. At the 7500 ppm dietary concentration, offspring bodyweight weight on post-natal days 1 and 4, offspring body weight gain to post-natal day 4 and litter weight on post-natal day 4 were lower than control. Offspring performance during assessment of surface righting was also inferior to control at the intermediate exposure level. At the 3000 ppm dietary concentration, offspring body weights, litter weights, offspring body weight gain and surface righting ability of the offspring were considered to be unaffected by maternal dietary exposure to the test material.

 

At the 7500 and 18000/12000 ppm dietary concentrations, there was a slightly higher incidence of clinical signs among offspring but these were concentrated in only a few litters (no milk visible in stomach (2 and 3 litters at 7500 ppm and 18000/12000 ppm respectively), cold (1 litter at 18000/12000 ppm), weak (1 litter at 18000/12000 ppm), small (2 and 3 litters at 7500 ppm and 18000/12000 ppm respectively) and distended abdomen (1 litter at 7500 ppm)), and did not indicate a clear effect of maternal dietary exposure on offspring development.

 

Based on the results this study the No Observed Adverse Effect Level (NOAEL) for systemic toxicity was considered to be 3000 ppm, principally due to effects on body weight gain, food consumption and adverse histopathological changes in the kidney and urinary bladder at 7500 ppm and 18000/12000 ppm.

 

The NOAEL for reproduction was considered to be 7500 ppm due to lower corpora lutea count at 18000/12000 ppm.

 

The NOAEL for survival, growth and development of the offspring was considered to be 3000 ppm due to effects on body weight, body weight gain and surface righting ability at 7500 ppm and 18000/12000 ppm.