Registration Dossier

Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015-07-28 to 2016-12-02
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:
sub-chronic toxicity: oral
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
2015-07-28 to 2016-12-02
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):
No clinical signs were apparent during the study in animals exposed to diets containing 2500, 5000 or 7500 ppm of the test item.
Mortality:
no mortality observed
Description (incidence):
There were no unscheduled deaths.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Animals of either sex exposed to 7500 ppm showed a statistically significant reduction in body weight gain during the first week of treatment. Recovery was evident in males thereafter, however a statistically significant reduction in body weight gain was evident in these males during Week 11 and overall weight gain was reduced by 6%. Females continued to show reduced body weight gain during Weeks 2, 3, 4, 6 and 10, with statistical significance being achieved during Weeks 4 and 6. Overall body weight gain for 7500 ppm group females was statistically significantly lower (29%) compared with controls. Statistically significantly lower mean body weight compared to controls was apparent in 7500 ppm group females from Day 15 until termination.

Overall body weight gain for males exposed to 5000 ppm and females exposed to 2500 and 5000 ppm was slightly lower than the control group and occasional statistically significant reductions in body weight (5000 ppm females only) and body weight gain for these animals relative to control were also noted. However, these differences did not show an exposure related response and were therefore considered to be unrelated to test item exposure.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
There was no adverse effect of dietary exposure to 2500, 5000 or 7500 ppm of the test item on food consumption for either sex or food conversion efficiency for males.

Reduced food conversion efficiency was evident in females exposed to 7500 ppm and followed the fluctuations seen in body weight gains for these females.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
There was no observed effect of dietary exposure to 2500, 5000 or 7500 ppm of the test item on water consumption for either sex.
Ophthalmological findings:
no effects observed
Description (incidence and severity):
Ophthalmic examination of the eyes from rats receiving diet containing 7500 ppm did not indicate any effect of treatment.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Animals of either sex fed diet containing 7500 ppm showed a statistically significant reduction in haemoglobin and haematocrit. Females exposed to 7500 ppm also showed a statistically significant reduction in erythrocyte count.

Females exposed to 5000 ppm showed a statistically significant reduction in hematocrit and haemoglobin. All of the individual values were within historical control ranges for rats of the strain and age used. In the absence of any associated histopathology correlates in the bone marrow or spleen, the intergroup differences were not considered to be of toxicological importance.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
There were no toxicologically significant effects detected in the blood chemical parameters examined.

Males from all dietary groups showed a statistically significant increase in bilirubin and a statistically significant reduction in bile acids. Females from all dietary groups showed a statistically significant increase in urea and a statistically significant reduction in alanine aminotransferase. Males fed diets containing 7500 ppm showed a statistically significant increase in cholesterol. Females fed diet containing 7500 ppm showed statistically significant reductions in glucose, total protein, albumin and bile acids and statistically significant increases in bilirubin and chloride concentration. The effect on total protein, albumin, bilirubin and bile acids extended to females fed diet containing 5000 ppm. Females fed diet containing 5000 ppm also showed a statistically significant increase in creatinine and a statistically significant reduction in phosphorus. Males fed diet containing 5000 ppm showed a statistically significant increase in cholesterol and a statistically significant reduction in albumin. Females fed diet containing 2500 ppm showed a statistically significant increase in creatinine. Individual values were within the historical control ranges and in the absence of exposure related responses (excluding bilirubin for females) or any associated histopathological correlates, the intergroup differences were not considered to be toxicologically significant.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Behavioural Assessments
Weekly assessment of the animals in a standard arena did not reveal any obvious adverse effects of dietary exposure to 2500, 5000 or 7500 ppm of the test item.

Functional Performance Tests
There were no toxicologically significant effects detected in functional performance.

Females exposed to 7500 ppm showed a statistically significant increase in hindlimb grip strength (two out of the three tests, p<0.05 or p<0.01). Females exposed to 7500 and 2500 ppm also showed a statistically significant increase in forelimb grip strength (one out of the three tests p<0.01). There was generally no dose-relationship and in the absence of any supporting clinical observations or pathological change to suggest a neurotoxic effect, the intergroup differences were considered to be of no toxicological significance.

Sensory Reactivity Assessments
There were no differences observed in the scores for sensory reactivity for either sex during the study.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Females exposed to diets containing 7500 and 5000 ppm showed a statistically significant and exposure related reduction in absolute adrenal weights. However, although relative (to terminal body weight) adrenal weights of females in the 7500 and 5000 ppm groups were statistically significantly lower than control, they did not follow a treatment related response. The majority of individual values for both absolute and relative weights for treated females were within historical control ranges, whereasindividual values for two control females exceeded the historical absolute ranges and three control females exceeded the historical control range for relative adrenal weight.

There was considered to be no effect of dietary exposure in 7500 or 5000 ppm males or in 2500 ppm animals of either sex on the organ weights measured.

In males fed diets containing 7500 ppm absolute and relative liver, brain and kidney weights were statistically significantly increased relative to control. The majority of the individual values were within the historical control range and in the absence of any histopathological correlates these intergroup differences were considered of no toxicological significance. Females fed diet containing 7500 and 5000 ppm showed a statistically significant reduction in absolute and relative (to terminal body weight) uterus/cervix weight but without any relationship to exposure. The reduction in the weight in the uterus/cervix in 7500 ppm group females was considered to be due to the stage of the reproductive cycle (fewer animals in proestrus/estrus) and of no toxicological significance.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Neither the type, incidence nor distribution of findings observed at terminal necropsy indicated any obvious effect of dietary exposure to 2500, 5000 or 7500 ppm of the test item. Incidences of reddened lungs were noted in a number of control and test item exposed
animals at necropsy and small testes and epididymides were evident in one male in the 5000 ppm group. These were all considered to be incidental findings.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
In the adrenals, mild hypertrophy of the zona glomerulosa was present in four males and four females fed diets containing 7500 ppm of the test item, and in two males and one female fed diets containing 5000 ppm. Hypertrophy of the zona glomerulosa is generally considered to be an adaptive process, usually linked to maintenance of electrolytes and fluid balance (Domenici Lombardo, 1990 and Greaves 2007). In this study, taking the blood chemistry values into account, changes in the zona glomerulosa of the adrenal gland were not considered to have affected normal electrolyteregulation. The histopathological changes in the adrenal glands at 7500 and 5000 ppm did not appear to have an effect on homeostatic control, and therefore were not considered to have had adverse consequences for the organ or the body. No similar effects were detected in animals of either sex fed diet containing 2500 ppm of the test item.

No other findings were present at histopathology which correlated with in-life changes noted. In particular the apparent reduction in weightin the uterus/cervix in high dose females was considered likely to be due to the stage of the reproductive cycle (fewer animals in proestrus/estrus) and of no toxicological significance.
Histopathological findings: neoplastic:
no effects observed
Other effects:
not examined
Dose descriptor:
NOAEL
Effect level:
5 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Systemic Toxicity
Critical effects observed:
no

Table 7. Group Mean Body Weight Gains – Male Rats

 

 

 

Group

 

Increase in Body Weight (g)

 

Day Numbers Relative to Start Date

Abs Gain

% Gain

From

1

8

15

22

29

36

43

50

57

64

71

78

85

1

1

To

8

15

22

29

36

43

50

57

64

71

78

85

91

91

91

 

Control

(0 ppm)

Mean

34.9

23.8

23.6

16.8

13.7

15.3

10.5

10.5

8.4

4.5

12.3

2.3

7.0

183.6

99.3

S.D.

7.5

3.9

5.0

5.5

4.9

7.5

2.8

5.7

4.5

3.0

4.4

3.5

3.1

39.7

21.5

N

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

 

Low Concn

(2500 ppm)

Mean

37.3

20.6

22.3

17.1

16.9

16.1

13.1

12.4

9.9

6.8

11.1

5.4

4.0

193.0

103.5

S.D.

3.5

3.8

6.5

4.5

9.3

9.7

3.9

3.6

4.1

2.6

4.7

3.5

4.7

28.9

16.6

N

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

 

Intermediate

Concn

(5000 ppm)

Mean

30.9

20.3

18.2

11.0*

14.0

13.7

11.6

13.7

9.5

6.9

7.5*

6.6*

5.3

169.2

91.6

S.D.

5.7

5.2

5.5

5.6

3.6

3.6

4.6

2.1

3.1

3.4

4.5

2.3

4.3

25.9

15.1

N

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

 

High Concn

(7500 ppm)

Mean

27.9**

21.5

18.1

13.2

17.7*

17.3

12.5

13.9

8.3

5.7

7.3*

3.8

5.8

173.0

95.0

S.D.

4.6

5.9

7.1

5.6

19.5

18.0

3.7

4.6

4.7

2.7

4.3

3.9

3.0

26.0

15.8

N

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

Table 8. Group Mean Body Weight Gains – Female Rats

 

 

 

Group

 

Increase in Body Weight (g)

 

Day Numbers Relative to Start Date

Abs Gain

% Gain

From

1

8

15

22

29

36

43

50

57

64

71

78

85

1

1

To

8

15

22

29

36

43

50

57

64

71

78

85

91

91

91

 

Control

(0 ppm)

Mean

14.3

14.5

9.2

7.7

4.5

7.4

3.9

5.2

2.2

4.1

3.1

0.5

3.1

79.7

51.0

S.D.

5.8

1.9

3.7

3.0

3.3

2.8

2.3

2.8

4.0

3.9

5.0

2.8

4.5

15.3

8.8

N

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

 

Low Concn

(2500 ppm)

Mean

11.9

12.3

10.4

5.8*

7.4

2.8*

4.1

4.8

2.6

2.1

4.6

0.6

-0.7

68.7

44.3

S.D.

3.9

3.6

3.4

2.0

3.4

4.5

2.7

2.6

3.9

2.1

2.9

2.0

3.3

9.8

7.3

N

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

 

Intermediate

Concn

(5000 ppm)

Mean

11.8

13.1

8.6

4.8*

6.4

6.0*

3.7

4.8

4.2

1.6

-1.5*

1.8

0.5

65.8*

43.7

S.D.

3.9

2.6

3.7

2.7

3.2

2.2

4.5

2.1

3.0

3.5

3.0

2.1

4.0

7.6

5.7

N

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

 

High Concn

(7500 ppm)

Mean

7.2**

11.8

7.0

5.3*

5.9

2.5**

3.7

5.5

2.1

1.7

2.4

0.9

0.5

56.5***

37.2

S.D.

7.8

6.1

4.8

2.1

3.3

1.6

3.5

3.6

2.3

2.6

2.7

3.4

4.5

10.5

7.3

N

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

Table 9. Group Mean Body Weight Values – Female Rats

Group

Body weights (g)

Day Numbers Relative to Start Date

From

1

8

15

22

29

36

43

50

57

64

71

78

85

91

Control

(0 ppm)

Mean

155.7

170.1

184.6

193.8

201.5

206.0

213.4

217.3

222.5

224.7

228.8

231.9

232.4

235.5

S.D.

7.0

10.3

10.2

11.7

13.8

16.4

15.3

15.1

16.2

18.9

19.9

16.9

18.0

19.9

N

10

10

10

10

10

10

10

10

10

10

10

10

10

10

Low Concn

(2500 ppm)

Mean

155.9

167.8

180.1

190.5

196.3

203.7

206.5

210.6

215.4

218.0

220.1

224.7

225.3

224.6

S.D.

9.0

10.0

8.8

7.6

7.6

7.9

9.3

8.0

9.2

11.5

11.8

11.4

11.3

12.0

N

10

10

10

10

10

10

10

10

10

10

10

10

10

10

Intermediate

Concn

(5000 ppm)

Mean

151.2

163.0

176.1

184.7

189.5

195.9

201.9

205.6

210.4

214.6

216.2

214.7*

216.5

217.0*

S.D.

8.0

7.0

8.1

11.3

10.4

9.8

9.4

12.2

12.0

10.3

12.1

12.0

11.7

10.3

N

10

10

10

10

10

10

10

10

10

10

10

10

10

10

High Concn

(7500 ppm)

Mean

152.1

159.3

171.1

*

178.1

*

183.4

**

189.3*

191.8

**

195.5

**

201.0

**

203.1

**

204.8

**

207.2

**

208.1

**

208.6

**

S.D.

9.0

12.2

13.5

17.0

16.5

16.2

15.9

16.3

15.6

15.3

16.2

15.6

16.2

13.9

N

10

10

10

10

10

10

10

10

10

10

10

10

10

10

Conclusions:
The continuous dietary administration of the test material to rats, at dietary concentrations of 2500, 5000 and 7500 ppm for ninety consecutive days, resulted in reduced body weight gains in animals of either sex exposed to 7500 ppm and microscopic adrenal changes in animals of either sex exposed to 7500 and 5000 ppm. The No Observed Effect Level (NOEL) for both sexes was therefore considered to be 2500 ppm (equivalent to a mean achieved dosage of 174.1 mg/kg bw/day for males and 196.4 mg/kg bw/day for females).
 
The microscopic adrenal changes (hypertrophy of the zona glomerulosa) identified in animals of either sex exposed to 7500 or 5000 ppm were mild. The changes were not considered to have affected normal electrolyte regulation and did not appear to have an effect on homeostatic control. Therefore, they were not considered to have had adverse consequences for the organ or the body. For this reason, 5000 ppm (equivalent to a mean achieved dosage
of 335.2 mg/kg bw/day for males and 401.2 mg/kg bw/day for females) was considered to be the “No Observed Adverse Effect Level” (NOAEL) systemic toxicity.
Executive summary:

In a key oral repeat dose toxicity study, the test material (Rosin, CAS# 8050-09-7) was administered continuously in the diet of three groups, each composed of ten male and ten female Wistar Han™:RccHan™:WIST strain rats, for ninety consecutive days, at dietary concentrations of 2500, 5000 or 7500 ppm (equivalent to a mean achieved dosage of 174.1, 335.2 or 510.1 mg/kg bw/day for males and 196.4, 401.2 or 596.2 mg/kg bw/day for females). A control group of ten males and ten females were fed basal laboratory diet.

 

Clinical signs, functional observations, body weight change, dietary intake and water consumption were monitored during the study. Haematology and blood chemistry were evaluated for all animals at the end of the study. Ophthalmoscopic examination was also performed on control group and high dose animals before the start of treatment and during Week 12 of the study. All animals were subjected to gross necropsy examination and a comprehensive

histopathological evaluation of tissues was performed.

 

No mortality was observed through the study period. There were no clinical signs observed that indicated any adverse effects of dietary exposures

at 2500, 5000 or 7500 ppm. Behavioural, functional, and sensory reactivity parameters remained unaffected by exposure to the test material and there were no adverse treatment-related effects on the eyes, water consumption, hematological, or clinical chemistry parameters evaluated. Gross necropsy did not reveal any remarkable findings.

 

Animals of either sex exposed to diets containing 7500 ppm showed a reduction in body weight gain during the first week of treatment. Recovery was evident in males thereafter, however a reduction in body weight gain was evident in these males during Week 11 and overall body weight gain in males was reduced relative to controls. Females continued to show a reduced body weight gain during Weeks 2, 3, 4, 6 and 10. Overall body weight gain for these females was reduced when compared to controls. Reduced food conversion efficiency was evident in females exposed to diets containing 7500 ppm, and followed the fluctuations seen in body weight gains for these females. No toxicologically significant effects on body weight were detected in animals of either sex exposed to diets containing 5000 or 2500 ppm.

 

Females exposed to diets containing 7500 and 5000 ppm showed a statistically significant reduction in adrenal weights, both absolute and relative to terminal body weight. However, relative adrenal weights of females in the 7500 and 5000 ppm groups did not follow an exposure related response and a majority of individual values for both absolute and relative weights for treated females were within historical control ranges. There was considered to be no effect of dietary exposure in 7500 or 5000 ppm males or in 2500 ppm animals of either sex on the organ weights measured.

 

Histopathological evaluation revealed hypertrophy of the zona glomerulosa in four males and four females exposed to 7500 ppm and in two males and one female exposed to 5000 ppm (all at a mild level). No such effects were detected in animals of either sex exposed to 2500 ppm. Hypertrophy of the zona glomerulosa is generally considered to be an adaptive or reactive change rather than an adverse effect of treatment (Domenici Lombardo, 1990 and Greaves, 2007) and was therefore not considered to be treatment-related.

 

The continuous dietary administration of the test material to rats, at dietary concentrations of 2500, 5000 and 7500 ppm for ninety consecutive days, resulted in reduced body weight gains in animals of either sex exposed to 7500 ppm and microscopic adrenal changes in animals of either sex exposed to 7500 and 5000 ppm. The No Observed Effect Level (NOEL) for both sexes was therefore considered to be 2500 ppm (equivalent to a mean achieved dosage of 174.1 mg/kg bw/day for males and 196.4 mg/kg bw/day for females).

 

The microscopic adrenal changes (hypertrophy of the zona glomerulosa) identified in animals of either sex exposed to 7500 or 5000 ppm were mild. The changes were not considered to have affected normal electrolyte regulation and did not appear to have an effect on homeostatic control. Therefore, they were not considered to have had adverse consequences for the organ or the body. For this reason, 5000 ppm (equivalent to a mean achieved dosage

of 335.2 mg/kg bw/day for males and 401.2 mg/kg bw/day for females) was considered to be the “No Observed Adverse Effect Level” (NOAEL) systemic toxicity.

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 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
yes
Remarks:
Deviations had no adverse impact on the scientific purpose of the study.
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Han™:RccHan™:WIST strain
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 UK Limited (Oxon, UK)
- Females (if applicable) nulliparous and non-pregnant: not specified
- Age at study initiation: 6-8 weeks
- Weight at study initiation: Males: 167 to 208g; Females: 141 to 170g
- Fasting period before study: Not specified
- Housing: groups of up to four by sex in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK).
- Diet (e.g. ad libitum): Ground diet (Rat and Mouse SQC Ground Diet No. 1, Special Diet Services, Dietex International Limited, Witham, Essex, UK) was used ad libitum
- Water (e.g. ad libitum): Mains drinking water was supplied ad libitum from polycarbonate bottles attached to the cage
- Acclimation period: 8 days

DETAILS OF FOOD AND WATER QUALITY: The diet, drinking water, bedding and environmental enrichment was considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.

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 twelvehours darkness

IN-LIFE DATES: From: 2015-08-21 To: 2015-11-20

Administration / exposure

Route of administration:
oral: feed
Details on route of administration:
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 considered to be relevant for the evaluation of the toxicological properties of the test item.
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): Dietary admixtures were prepared fortnightly and stored at room temperature
- Mixing appropriate amounts with (Type of food): A known amount of test item was mixed with a small amount of basal laboratory diet in a Robot Coupe Blixer 4 mixer until homogeneous.This pre-mix was then added to a larger amount of basal laboratory diet and mixed for a further sixty minutes at a constant speed, setting 1 in a Hobart H800 mixer.
- Storage temperature of food: stable for 20 days at room temperature
:
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Representative samples of dietary admixtures were taken and analyzed for concentration of Rosin CAS 8050-09-7 at Envigo Research Limited, Shardlow, UK, Analytical Services. The results indicate that the prepared formulations were within acceptable limits of the nominal concentration, thus confirming the suitability and accuracy of the formulation procedure.
Duration of treatment / exposure:
ninety consecutive days
Frequency of treatment:
continuously in the diet, for ninety consecutive days
Doses / concentrationsopen allclose all
Dose / conc.:
0 ppm
Remarks:
Control
Dose / conc.:
2 500 ppm
Remarks:
Low Concentration
Dose / conc.:
5 000 ppm
Remarks:
Intermediate Concentration
Dose / conc.:
7 500 ppm
Remarks:
High Concentration
No. of animals per sex per dose:
10/sex/concentration
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: The dietary concentrations were chosen based on the results of a previous toxicity study (Harlan Laboratories Ltd., Study Number: D80926: Tall Oil Rosin - CAS no. 8050-09-7: Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test in the Han Wistar Rat).
- Rationale for animal assignment (if not random): The 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 cages were distributed in dose group columns within the holding rack to minimize the potentialof cross contamination of the treated diet. The animals were uniquely identified within the study by an ear punching system routinely used in these laboratories.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All animals were examined for overt signs of toxicity, ill-health or behavioural change daily from the start of treatment.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were recorded on Day 1 (prior the start of treatment) and at weekly intervals thereafter. 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
Food consumption was recorded for each cage group at weekly intervals throughout the study

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Not specified

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: Water intake was observed daily, for each cage group, by visual inspection of the water bottles for any overt changes

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: pre-treatment and before termination of treatment (during Week 12).
- Dose groups that were examined: all control and high dose animals

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Day 90
- Anaesthetic used for blood collection: Not specified
- Animals fasted: No
- How many animals: all animals from each test and control group
- Parameters checked in table [No.2] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Day 90
- Animals fasted: No
- How many animals: all animals from each test and control group
- Parameters checked in table [No.3] were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Prior to the start of treatment and at weekly intervals thereafter, all animals were observed for signs of functional/behavioural toxicity
- Dose groups that were examined: all animals
- Battery of functions tested: sensory activity / grip strength / motor activity
- Parameters checked in table [No.4] were examined.

IMMUNOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table 5.)

HISTOPATHOLOGY: Yes (see table 6.)
Statistics:
See "any other information on materials and methods incl. tables" for information on statistics.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
No clinical signs were apparent during the study in animals exposed to diets containing 2500, 5000 or 7500 ppm of the test item.
Mortality:
no mortality observed
Description (incidence):
There were no unscheduled deaths.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Animals of either sex exposed to 7500 ppm showed a statistically significant reduction in body weight gain during the first week of treatment. Recovery was evident in males thereafter, however a statistically significant reduction in body weight gain was evident in these males during Week 11 and overall weight gain was reduced by 6%. Females continued to show reduced body weight gain during Weeks 2, 3, 4, 6 and 10, with statistical significance being achieved during Weeks 4 and 6. Overall body weight gain for 7500 ppm group females was statistically significantly lower (29%) compared with controls. Statistically significantly lower mean body weight compared to controls was apparent in 7500 ppm group females from Day 15 until termination.

Overall body weight gain for males exposed to 5000 ppm and females exposed to 2500 and 5000 ppm was slightly lower than the control group and occasional statistically significant reductions in body weight (5000 ppm females only) and body weight gain for these animals relative to control were also noted. However, these differences did not show an exposure related response and were therefore considered to be unrelated to test item exposure.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
There was no adverse effect of dietary exposure to 2500, 5000 or 7500 ppm of the test item on food consumption for either sex or food conversion efficiency for males.

Reduced food conversion efficiency was evident in females exposed to 7500 ppm and followed the fluctuations seen in body weight gains for these females.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
There was no observed effect of dietary exposure to 2500, 5000 or 7500 ppm of the test item on water consumption for either sex.
Ophthalmological findings:
no effects observed
Description (incidence and severity):
Ophthalmic examination of the eyes from rats receiving diet containing 7500 ppm did not indicate any effect of treatment.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Animals of either sex fed diet containing 7500 ppm showed a statistically significant reduction in haemoglobin and haematocrit. Females exposed to 7500 ppm also showed a statistically significant reduction in erythrocyte count.

Females exposed to 5000 ppm showed a statistically significant reduction in hematocrit and haemoglobin. All of the individual values were within historical control ranges for rats of the strain and age used. In the absence of any associated histopathology correlates in the bone marrow or spleen, the intergroup differences were not considered to be of toxicological importance.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
There were no toxicologically significant effects detected in the blood chemical parameters examined.

Males from all dietary groups showed a statistically significant increase in bilirubin and a statistically significant reduction in bile acids. Females from all dietary groups showed a statistically significant increase in urea and a statistically significant reduction in alanine aminotransferase. Males fed diets containing 7500 ppm showed a statistically significant increase in cholesterol. Females fed diet containing 7500 ppm showed statistically significant reductions in glucose, total protein, albumin and bile acids and statistically significant increases in bilirubin and chloride concentration. The effect on total protein, albumin, bilirubin and bile acids extended to females fed diet containing 5000 ppm. Females fed diet containing 5000 ppm also showed a statistically significant increase in creatinine and a statistically significant reduction in phosphorus. Males fed diet containing 5000 ppm showed a statistically significant increase in cholesterol and a statistically significant reduction in albumin. Females fed diet containing 2500 ppm showed a statistically significant increase in creatinine. Individual values were within the historical control ranges and in the absence of exposure related responses (excluding bilirubin for females) or any associated histopathological correlates, the intergroup differences were not considered to be toxicologically significant.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Behavioural Assessments
Weekly assessment of the animals in a standard arena did not reveal any obvious adverse effects of dietary exposure to 2500, 5000 or 7500 ppm of the test item.

Functional Performance Tests
There were no toxicologically significant effects detected in functional performance.

Females exposed to 7500 ppm showed a statistically significant increase in hindlimb grip strength (two out of the three tests, p<0.05 or p<0.01). Females exposed to 7500 and 2500 ppm also showed a statistically significant increase in forelimb grip strength (one out of the three tests p<0.01). There was generally no dose-relationship and in the absence of any supporting clinical observations or pathological change to suggest a neurotoxic effect, the intergroup differences were considered to be of no toxicological significance.

Sensory Reactivity Assessments
There were no differences observed in the scores for sensory reactivity for either sex during the study.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Females exposed to diets containing 7500 and 5000 ppm showed a statistically significant and exposure related reduction in absolute adrenal weights. However, although relative (to terminal body weight) adrenal weights of females in the 7500 and 5000 ppm groups were statistically significantly lower than control, they did not follow a treatment related response. The majority of individual values for both absolute and relative weights for treated females were within historical control ranges, whereasindividual values for two control females exceeded the historical absolute ranges and three control females exceeded the historical control range for relative adrenal weight.

There was considered to be no effect of dietary exposure in 7500 or 5000 ppm males or in 2500 ppm animals of either sex on the organ weights measured.

In males fed diets containing 7500 ppm absolute and relative liver, brain and kidney weights were statistically significantly increased relative to control. The majority of the individual values were within the historical control range and in the absence of any histopathological correlates these intergroup differences were considered of no toxicological significance. Females fed diet containing 7500 and 5000 ppm showed a statistically significant reduction in absolute and relative (to terminal body weight) uterus/cervix weight but without any relationship to exposure. The reduction in the weight in the uterus/cervix in 7500 ppm group females was considered to be due to the stage of the reproductive cycle (fewer animals in proestrus/estrus) and of no toxicological significance.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Neither the type, incidence nor distribution of findings observed at terminal necropsy indicated any obvious effect of dietary exposure to 2500, 5000 or 7500 ppm of the test item. Incidences of reddened lungs were noted in a number of control and test item exposed
animals at necropsy and small testes and epididymides were evident in one male in the 5000 ppm group. These were all considered to be incidental findings.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
In the adrenals, mild hypertrophy of the zona glomerulosa was present in four males and four females fed diets containing 7500 ppm of the test item, and in two males and one female fed diets containing 5000 ppm. Hypertrophy of the zona glomerulosa is generally considered to be an adaptive process, usually linked to maintenance of electrolytes and fluid balance (Domenici Lombardo, 1990 and Greaves 2007). In this study, taking the blood chemistry values into account, changes in the zona glomerulosa of the adrenal gland were not considered to have affected normal electrolyteregulation. The histopathological changes in the adrenal glands at 7500 and 5000 ppm did not appear to have an effect on homeostatic control, and therefore were not considered to have had adverse consequences for the organ or the body. No similar effects were detected in animals of either sex fed diet containing 2500 ppm of the test item.

No other findings were present at histopathology which correlated with in-life changes noted. In particular the apparent reduction in weightin the uterus/cervix in high dose females was considered likely to be due to the stage of the reproductive cycle (fewer animals in proestrus/estrus) and of no toxicological significance.
Histopathological findings: neoplastic:
no effects observed
Other effects:
not examined

Effect levels

Dose descriptor:
NOAEL
Effect level:
5 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Systemic Toxicity

Target system / organ toxicity

Critical effects observed:
no

Any other information on results incl. tables

Table 7. Group Mean Body Weight Gains – Male Rats

 

 

 

Group

 

Increase in Body Weight (g)

 

Day Numbers Relative to Start Date

Abs Gain

% Gain

From

1

8

15

22

29

36

43

50

57

64

71

78

85

1

1

To

8

15

22

29

36

43

50

57

64

71

78

85

91

91

91

 

Control

(0 ppm)

Mean

34.9

23.8

23.6

16.8

13.7

15.3

10.5

10.5

8.4

4.5

12.3

2.3

7.0

183.6

99.3

S.D.

7.5

3.9

5.0

5.5

4.9

7.5

2.8

5.7

4.5

3.0

4.4

3.5

3.1

39.7

21.5

N

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

 

Low Concn

(2500 ppm)

Mean

37.3

20.6

22.3

17.1

16.9

16.1

13.1

12.4

9.9

6.8

11.1

5.4

4.0

193.0

103.5

S.D.

3.5

3.8

6.5

4.5

9.3

9.7

3.9

3.6

4.1

2.6

4.7

3.5

4.7

28.9

16.6

N

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

 

Intermediate

Concn

(5000 ppm)

Mean

30.9

20.3

18.2

11.0*

14.0

13.7

11.6

13.7

9.5

6.9

7.5*

6.6*

5.3

169.2

91.6

S.D.

5.7

5.2

5.5

5.6

3.6

3.6

4.6

2.1

3.1

3.4

4.5

2.3

4.3

25.9

15.1

N

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

 

High Concn

(7500 ppm)

Mean

27.9**

21.5

18.1

13.2

17.7*

17.3

12.5

13.9

8.3

5.7

7.3*

3.8

5.8

173.0

95.0

S.D.

4.6

5.9

7.1

5.6

19.5

18.0

3.7

4.6

4.7

2.7

4.3

3.9

3.0

26.0

15.8

N

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

Table 8. Group Mean Body Weight Gains – Female Rats

 

 

 

Group

 

Increase in Body Weight (g)

 

Day Numbers Relative to Start Date

Abs Gain

% Gain

From

1

8

15

22

29

36

43

50

57

64

71

78

85

1

1

To

8

15

22

29

36

43

50

57

64

71

78

85

91

91

91

 

Control

(0 ppm)

Mean

14.3

14.5

9.2

7.7

4.5

7.4

3.9

5.2

2.2

4.1

3.1

0.5

3.1

79.7

51.0

S.D.

5.8

1.9

3.7

3.0

3.3

2.8

2.3

2.8

4.0

3.9

5.0

2.8

4.5

15.3

8.8

N

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

 

Low Concn

(2500 ppm)

Mean

11.9

12.3

10.4

5.8*

7.4

2.8*

4.1

4.8

2.6

2.1

4.6

0.6

-0.7

68.7

44.3

S.D.

3.9

3.6

3.4

2.0

3.4

4.5

2.7

2.6

3.9

2.1

2.9

2.0

3.3

9.8

7.3

N

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

 

Intermediate

Concn

(5000 ppm)

Mean

11.8

13.1

8.6

4.8*

6.4

6.0*

3.7

4.8

4.2

1.6

-1.5*

1.8

0.5

65.8*

43.7

S.D.

3.9

2.6

3.7

2.7

3.2

2.2

4.5

2.1

3.0

3.5

3.0

2.1

4.0

7.6

5.7

N

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

 

High Concn

(7500 ppm)

Mean

7.2**

11.8

7.0

5.3*

5.9

2.5**

3.7

5.5

2.1

1.7

2.4

0.9

0.5

56.5***

37.2

S.D.

7.8

6.1

4.8

2.1

3.3

1.6

3.5

3.6

2.3

2.6

2.7

3.4

4.5

10.5

7.3

N

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

Table 9. Group Mean Body Weight Values – Female Rats

Group

Body weights (g)

Day Numbers Relative to Start Date

From

1

8

15

22

29

36

43

50

57

64

71

78

85

91

Control

(0 ppm)

Mean

155.7

170.1

184.6

193.8

201.5

206.0

213.4

217.3

222.5

224.7

228.8

231.9

232.4

235.5

S.D.

7.0

10.3

10.2

11.7

13.8

16.4

15.3

15.1

16.2

18.9

19.9

16.9

18.0

19.9

N

10

10

10

10

10

10

10

10

10

10

10

10

10

10

Low Concn

(2500 ppm)

Mean

155.9

167.8

180.1

190.5

196.3

203.7

206.5

210.6

215.4

218.0

220.1

224.7

225.3

224.6

S.D.

9.0

10.0

8.8

7.6

7.6

7.9

9.3

8.0

9.2

11.5

11.8

11.4

11.3

12.0

N

10

10

10

10

10

10

10

10

10

10

10

10

10

10

Intermediate

Concn

(5000 ppm)

Mean

151.2

163.0

176.1

184.7

189.5

195.9

201.9

205.6

210.4

214.6

216.2

214.7*

216.5

217.0*

S.D.

8.0

7.0

8.1

11.3

10.4

9.8

9.4

12.2

12.0

10.3

12.1

12.0

11.7

10.3

N

10

10

10

10

10

10

10

10

10

10

10

10

10

10

High Concn

(7500 ppm)

Mean

152.1

159.3

171.1

*

178.1

*

183.4

**

189.3*

191.8

**

195.5

**

201.0

**

203.1

**

204.8

**

207.2

**

208.1

**

208.6

**

S.D.

9.0

12.2

13.5

17.0

16.5

16.2

15.9

16.3

15.6

15.3

16.2

15.6

16.2

13.9

N

10

10

10

10

10

10

10

10

10

10

10

10

10

10

Applicant's summary and conclusion

Conclusions:
The continuous dietary administration of the test material to rats, at dietary concentrations of 2500, 5000 and 7500 ppm for ninety consecutive days, resulted in reduced body weight gains in animals of either sex exposed to 7500 ppm and microscopic adrenal changes in animals of either sex exposed to 7500 and 5000 ppm. The No Observed Effect Level (NOEL) for both sexes was therefore considered to be 2500 ppm (equivalent to a mean achieved dosage of 174.1 mg/kg bw/day for males and 196.4 mg/kg bw/day for females).
 
The microscopic adrenal changes (hypertrophy of the zona glomerulosa) identified in animals of either sex exposed to 7500 or 5000 ppm were mild. The changes were not considered to have affected normal electrolyte regulation and did not appear to have an effect on homeostatic control. Therefore, they were not considered to have had adverse consequences for the organ or the body. For this reason, 5000 ppm (equivalent to a mean achieved dosage
of 335.2 mg/kg bw/day for males and 401.2 mg/kg bw/day for females) was considered to be the “No Observed Adverse Effect Level” (NOAEL) systemic toxicity.
Executive summary:

In a key oral repeat dose toxicity study, the test material (Rosin, CAS# 8050-09-7) was administered continuously in the diet of three groups, each composed of ten male and ten female Wistar Han™:RccHan™:WIST strain rats, for ninety consecutive days, at dietary concentrations of 2500, 5000 or 7500 ppm (equivalent to a mean achieved dosage of 174.1, 335.2 or 510.1 mg/kg bw/day for males and 196.4, 401.2 or 596.2 mg/kg bw/day for females). A control group of ten males and ten females were fed basal laboratory diet.

 

Clinical signs, functional observations, body weight change, dietary intake and water consumption were monitored during the study. Haematology and blood chemistry were evaluated for all animals at the end of the study. Ophthalmoscopic examination was also performed on control group and high dose animals before the start of treatment and during Week 12 of the study. All animals were subjected to gross necropsy examination and a comprehensive

histopathological evaluation of tissues was performed.

 

No mortality was observed through the study period. There were no clinical signs observed that indicated any adverse effects of dietary exposures

at 2500, 5000 or 7500 ppm. Behavioural, functional, and sensory reactivity parameters remained unaffected by exposure to the test material and there were no adverse treatment-related effects on the eyes, water consumption, hematological, or clinical chemistry parameters evaluated. Gross necropsy did not reveal any remarkable findings.

 

Animals of either sex exposed to diets containing 7500 ppm showed a reduction in body weight gain during the first week of treatment. Recovery was evident in males thereafter, however a reduction in body weight gain was evident in these males during Week 11 and overall body weight gain in males was reduced relative to controls. Females continued to show a reduced body weight gain during Weeks 2, 3, 4, 6 and 10. Overall body weight gain for these females was reduced when compared to controls. Reduced food conversion efficiency was evident in females exposed to diets containing 7500 ppm, and followed the fluctuations seen in body weight gains for these females. No toxicologically significant effects on body weight were detected in animals of either sex exposed to diets containing 5000 or 2500 ppm.

 

Females exposed to diets containing 7500 and 5000 ppm showed a statistically significant reduction in adrenal weights, both absolute and relative to terminal body weight. However, relative adrenal weights of females in the 7500 and 5000 ppm groups did not follow an exposure related response and a majority of individual values for both absolute and relative weights for treated females were within historical control ranges. There was considered to be no effect of dietary exposure in 7500 or 5000 ppm males or in 2500 ppm animals of either sex on the organ weights measured.

 

Histopathological evaluation revealed hypertrophy of the zona glomerulosa in four males and four females exposed to 7500 ppm and in two males and one female exposed to 5000 ppm (all at a mild level). No such effects were detected in animals of either sex exposed to 2500 ppm. Hypertrophy of the zona glomerulosa is generally considered to be an adaptive or reactive change rather than an adverse effect of treatment (Domenici Lombardo, 1990 and Greaves, 2007) and was therefore not considered to be treatment-related.

 

The continuous dietary administration of the test material to rats, at dietary concentrations of 2500, 5000 and 7500 ppm for ninety consecutive days, resulted in reduced body weight gains in animals of either sex exposed to 7500 ppm and microscopic adrenal changes in animals of either sex exposed to 7500 and 5000 ppm. The No Observed Effect Level (NOEL) for both sexes was therefore considered to be 2500 ppm (equivalent to a mean achieved dosage of 174.1 mg/kg bw/day for males and 196.4 mg/kg bw/day for females).

 

The microscopic adrenal changes (hypertrophy of the zona glomerulosa) identified in animals of either sex exposed to 7500 or 5000 ppm were mild. The changes were not considered to have affected normal electrolyte regulation and did not appear to have an effect on homeostatic control. Therefore, they were not considered to have had adverse consequences for the organ or the body. For this reason, 5000 ppm (equivalent to a mean achieved dosage

of 335.2 mg/kg bw/day for males and 401.2 mg/kg bw/day for females) was considered to be the “No Observed Adverse Effect Level” (NOAEL) systemic toxicity.