Registration Dossier

Toxicological information

Repeated dose toxicity: oral

Currently viewing:

Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015-10-13 to 2016-04-25
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 for cross-reference:
read-across: supporting information

Data source

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

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
yes
Remarks:
Deviation did not impact the integrity of the study
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Reference substance name:
Rosin, reaction products with formaldehyde
EC Number:
293-659-0
EC Name:
Rosin, reaction products with formaldehyde
Details on test material:
- Name of test material (as cited in study report): Rosin, reaction product with formaldehyde
- CAS No. 91081-53-7
- Substance type: UVCB
- Physical state: Brownish solid
- Purity/Concentration as Supplied: 99.72%
- Purity/Concentration for Formulations: 100%
- Correction for Purity: No
- Lot/batch No.:MH131391
- Stability of Test Item in Feed Preparation: At least 8 days if stored at room temperature (20 ± 5 °C) in the dark.
- Expiration date of the lot/batch: 27-Jun-2014
- Storage conditions: Bulk sample was stored frozen (-20 ± 5 °C) under nitrogen in the dark. For temperature adjustment, working samples were kept under nitrogen at room temperature (20 ± 5 °C) for a maximum of 3 hours, prior to feed preparation.
- Safety Precautions: Routine hygienic procedures (gloves, goggles, face mask).
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Hydrocarbons Resins and Rosin Resins REACH Consortium (c/o Penman Consulting BVBA, Rue Royale 157, Bte 13 B-1210 Brussels (Saint-Josse-ten-Noode), BELGIUM); Batch No. MH151758
- Expiration date of the lot/batch: 2016-08-03
- Purity test date: 2015-08-03

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Approximately -20°C, under Nitrogen, in the dark
- Stability under test conditions:Not specified

FORM AS APPLIED IN THE TEST (if different from that of starting material): Amber coloured solid

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 or test system and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS (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: 187 to 234 g; Females: 149 to 183 g
- Fasting period before study: Not specified
- Housing: Housed in 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, Envigo RMS (UK) Limited, Oxon, 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: 7 days

DETAILS OF FOOD AND WATER QUALITY: Environmental enrichment was provided in the form of wooden chew blocks and cardboard fun tunnels (Datesand Ltd., Cheshire, UK). 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: 2015-10-16 To: 2016-01-15

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 believed to be of value in assessing the toxicological properties of the test item.
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): Diets were prepared in approximately weekly mixes
- Mixing appropriate amounts with (Type of food): A known amount of test item was mixed with a small amount of basal laboratory diet until considered to be homogeneous at a constant speed, setting 1 in a Robot Couple Blixer 4 mixer. 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 or Hobart U200 mixer.
- Storage temperature of food: stored at approximately -20°C from preparation until their first day of allocation. Thereafter they were stored at ambient temperature in the dark until completion of the allocated week of treatment, when they were discarded.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples were taken of each dietary admixture and analysed for concentration of Rosin, reaction products with formaldehyde CAS 91081-53-7 at Envigo Research Limited, Shardlow, UK, Analytical Services. The results indicate that the prepared dietary
admixture concentrations were within ± 93 to 115% of the nominal concentration and indicated that diets were in acceptable ranges for the purpose of this study.
Duration of treatment / exposure:
90 days
Frequency of treatment:
Continuously, for ninety consecutive days, by dietary admixture.
Doses / concentrationsopen allclose all
Dose / conc.:
0 ppm
Remarks:
Control
Dose / conc.:
1 000 ppm
Remarks:
Low Concentration
Dose / conc.:
3 000 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 previous toxicity studies, including an OECD 422 study (Harlan Laboratories, Switzerland, study number D81016).
- Rationale for animal assignment (if not random): The animals were randomly allocated to treatment groups using a stratified body weight randomisation 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 randomised. 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. All observations were recorded

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: Yes

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
- Dose groups that were examined: all animals
- Battery of functions tested: sensory activity / grip strength / motor activity / other: - Parameters checked in table [No. 4] were examined.

IMMUNOLOGY: No
Sacrifice and pathology:
On completion of the dosing period all animals were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination. All animals were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

GROSS PATHOLOGY: Yes (see table 5)

HISTOPATHOLOGY: Yes (see table 6)
Statistics:
Where considered appropriate, quantitative data was subjected to statistical analysis to detect the significance of intergroup differences from control; statistical significance was achieved at a level of p<0.05. Statistical analysis was performed on the following parameters:

Grip Strength, Motor Activity, Body Weight, Body Weight Change, Hematology, Blood Chemistry, Absolute Organ Weights, Body Weight-Relative Organ Weights.

Data were analysed using the decision tree from the ProvantisTM Tables and Statistics Module as detailed as follows:
Where appropriate, data transformations were performed using the most suitable method. The homogeneity of variance from mean values was analysed using Bartlett’s test. Intergroup variances were assessed using suitable ANOVA, or if required, ANCOVA with appropriate covariates. Any transformed data were analysed to find the lowest treatment level that showed a significant effect using the Williams Test for parametric data or the Shirley Test for non-parametric data. If no dose response was found but the data shows nonhomogeneity of means, the data were analysed by a stepwise Dunnett’s (parametric) or Steel (non-parametric) test to determine significant difference from the control group. Where the data were unsuitable for these analyses, pair-wise tests was performed using the Student t-test (parametric) or the Mann-Whitney U test (non-parametric).

Probability values (p) are presented as follows:
p<0.01 **
p<0.05 *
p>0.05 (not significant)

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
The low incidence of clinical signs apparent during the study indicatedthat dietary exposure to 1000, 3000 or 7500 ppm of the test item produced no obvious clinical effects.
Mortality:
no mortality observed
Description (incidence):
There were no unscheduled deaths on the study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Females:
Females exposed to 7500 ppm of the test item showed statistically significant (p<0.01) lower mean body weight gain during the first week of the study compared with control. Body weight gains for these females remained lower, but not statistically significant during the following two weeks. Subsequent body weight gain was essentially similar to control until Week 6 (Days 36-43), when statistically significant (p<0.01) lower mean body weight gain was again apparent. No statistically significant differences in body weight gain were subsequently observed to termination, although lower body weight gains were apparent during Days 50-57 and 71-78 and higher body weight gains were seen during Days 57-64, 64-71 and 85-91. The lower initial body weight gain resulted in a statistically significant (p<0.05) lower mean body weight for Week 2 and mean body weight remained statistically significantly (p<0.05 - p<0.01) lower than control until termination. Overall body weight gain from the start of treatment to termination was also statistically significantly (p<0.01) lower than control.

Females exposed to 1000 or 3000 ppm of the test item also showed statistically significant (p<0.01) lower mean body weight gain during the first week of the study compared with control, but there was no relationship to dietary exposure of the test item. Thereafter, overall body weight gain from Week 1 to termination was similar to control, although occasionally weekly mean body weight gains were lower than control, but not statistically significant. Overall body weight gain and group mean body weight from the start of treatment was not statistically significantly different from control.Actual intergroup differences for females at 1000 and 3000 ppm of the test item was a mean of approximately 8 g at the beginning of the study and approximately 10.5 g by the end. This difference is considered insufficient evidence to conclude an exposure-related effect.

Males:
For males exposed to 1000, 3000 or 7500 ppm of the test item, statistically significant (p<0.05-p<0.01) lower body weight gains compared to control were apparent during Week 7 (Days 43-50).

Males exposed to 1000, 3000 or 7500 ppm of the test item showed statistically significant (p<0.05-p<0.01) lower body weight gain during Week 9 (Days 57-64) but statistically significant (p<0.01) higher body weight gain during Week 10 (64-71).

For males exposed to 1000 ppm of the test item, statistically significant (p<0.01) higher body weight gains compared to control were apparent during Week 4 (Days 15-22). Males exposed to 1000, 3000 or 7500 ppm of the test item showed no statistically significant differences from control for overall body weight gain or for mean body weights throughout the study.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Females:
Females exposed to 7500 ppm of the test item generally showed lower food consumption than control throughout the study with differences being most noticeable during the first two weeks (Days 1-15), Week 11 (Days 71-78) and final week (Days 85-91) of the study. Females exposed to 1000 or 3000 ppm of the test item showed lower food consumption compared to control during the first week of the study; food intake at both these exposures were lower than that observed for females exposed to 7500 ppm of the test item during this period. Food consumption of females exposed to 1000 or 3000 ppm of the test item was similar to control for the remainder of the study.

Males:
For males, intergroup differences in food consumption at dietary concentrations of 1000, 3000 and 7500 ppm of the test item did not reveal any obvious effect of treatment on food intake.
Food efficiency:
effects observed, non-treatment-related
Description (incidence and severity):
Females:
Females exposed to 7500 ppm of the test item showed lower food conversion efficiency, compared to control, during Days 1-8, 36-43 and 71-78 of the study. To a lesser extent, food conversion efficiency was lower than control during Days 8-22 and 50-57 and higher than control during Days 57-71 and Days 85-91 but differences did not indicate a clear association with treatment.

Males:
Intergroup differences in food conversion efficiency did not indicate any obvious consistent effect of dietary exposure for either sex exposed to 1000 or 3000 ppm of the test item or males exposed to 7500 ppm of the test item.
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
There was no observed effect of dietary exposure on water consumption for either sex at dietary concentrations of 1000, 3000 and 7500 ppm of the test item.
Ophthalmological findings:
no effects observed
Description (incidence and severity):
Ophthalmoscopic examinations of animals exposed to 7500 ppm of the test item did not indicate any effect of treatment.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Males:
Males exposed to 7500 ppm of the test item showed statistically significant (p<0.05) lower haemoglobin and haematocrit levels compared with control; for haemoglobin, the group mean value was also below the historical control range.

Males exposed to 7500 ppm of the test item showed a statistically significant (p<0.05) higher number of platelets compared with control,with the group mean value being above the historical control range.

Males exposed to 7500 ppm of the test item, showed statistically significant (p<0.01) longer prothrombin time compared to control. However, the group mean value at 7500 ppm was within the historical control range, while the group mean control value was outside the historical control range.

Females:
Females exposed to 1000, 3000 and 7500 ppm ofthe test item, showed statistically significant (p<0.05), lower total leukocyte count compared to control, principally due to a statistically significant lower number of lymphocytes. Group mean values showed no relationship to exposure and were all within the respective historical control data, while the control value for leukocyte count exceeded the historical range.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Males:
Males exposed to 1000, 3000 and 7500 ppm of the test item showed statistically significant (p<0.05) higher albumin/globulin ratio compared to control. Group mean values showed no relationship to exposure and were within the historical control range. There were no accompanying statistically significant differencesfrom control apparent for total protein or albumin level for males exposed to 1000, 3000 and 7500 ppm of the test item.

Females:
There were no statistically significant differencesfrom control for biochemistry parameters apparent for females exposed to 1000, 3000 and 7500 ppm of the test item.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Behavioural Assessments
Weekly assessment of the animals in a standard arena did not reveal any obvious effects of dietary exposure to 1000, 3000 or 7500 ppm of the test item.

Functional Performance Tests
For males exposed to 3000 or 7500 ppm of the test item, statistically significant (p<0.05) lower forelimb grip strength was apparent during trial 2, compared with the control. However, no similar statistically significant differences in forelimb grip strength were apparent during trials 1 and 3.

For males exposed to 3000 ppm of the test item, statistically significant (p<0.05) lower motor activity was observed during the last 20% of the measurement period. No similar effect was apparent for either sex at 7500 ppm.

For females exposed to 1000 ppm of the test item, statistically significant (p<0.01) lower overall motor activity was observed. No similar findings were apparentfor females exposed to higher dietary exposure levels.

Sensory Reactivity Assessments
Intergroup differences observed in the scores for sensory reactivity did not indicate any effect of treatment for either sex at dietary concentrations of 1000, 3000 or 7500 ppm of the test item.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Males exposed to 3000 and 7500 ppm of the testitem, showed statistically significant (p<0.05 and p<0.01 retrospectively) higher absolute and relative liver weight compared to control. For males exposed to 7500 ppm of the test item, the group mean relative liver weight exceeded the historical control range. The increases in liver weights observed were considered to be related to test item exposure.

No statistically significant differences in organ weights were apparent for males exposed to 1000 ppm of the test item or for females exposed to 1000, 3000 or 7500 ppm of the test item.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Macroscopic observations at terminal necropsy did not indicate any obvious effect of treatment for either sex exposed to 1000, 3000 or 7500 ppm of the test item.
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
LIVER: Centrilobular hepatocyte hypertrophy was noted in 4/10 males exposed to 7500 ppm of the test item.

THYROID GLAND : Follicular cell hypertrophy was present in 1/10 and 4/10 males exposed to 3000 and 7500 ppm of the test item respectively.

No histopathological findings considered to be related to exposure to the test item were identified in males at 1000 ppm and females at 1000, 3000 and 7500 ppm of the test item.

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
3 000 ppm
Based on:
test mat.
Sex:
female
Basis for effect level:
other: Systemic Toxicity
Remarks on result:
other: equivalent to mean achieved dosages of approximately 213.1 mg/kg bw/day (males) or 255.2 mg/kg bw/day (females)

Target system / organ toxicity

Key result
Critical effects observed:
no

Any other information on results incl. tables

Table 7. 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

165.8

182.7

193.0

201.4

210.7

218.3

224.4

229.2

235.6

238.4

239.3

246.3

247.4

250.9

S.D.

8.1

7.2

9.6

13.0

15.0

13.3

13.9

17.8

18.0

13.4

15.8

19.7

19.9

19.2

N

10

10

10

10

10

10

10

10

10

10

10

10

10

10

Low Concn

(1000 ppm)

Mean

169.7

181.0

191.2

202.1

211.7

216.4

220.9

225.4

230.6

233.0

235.8

238.6

243.3

249.6

S.D.

6.6

9.2

10.2

11.6

12.8

13.1

15.2

16.7

16.6

17.7

17.8

20.7

20.5

21.4

N

10

10

10

10

10

10

10

10

10

10

10

10

10

10

Intermediate

Concn

(3000 ppm)

Mean

161.8

174.0

183.7

194.0

202.3

210.7

214.3

219.3

223.8

228.1

232.1

231.5

234.4

239.1

S.D.

7.0

9.7

15.5

18.1

19.6

21.4

22.4

23.0

23.3

24.2

25.7

24.8

27.3

26.4

N

10

10

10

10

10

10

10

10

10

10

10

10

10

10

High Concn

(7500 ppm)

Mean

162.9

173.0*

179.0*

184.6*

193.7*

200.7*

201.4

**

205.0

**

208.3

**

213.3

**

217.3*

217.5

**

217.8

**

222.3

**

S.D.

5.0

6.8

7.6

8.2

9.1

7.4

7.2

8.0

8.6

7.9

7.9

9.1

9.2

9.4

N

10

10

10

10

10

10

10

10

10

10

10

10

10

10

*  Significantly different from control group p<0.05

**  Significantly different from control group p<0.01

Table 8. 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

29.3

26.4

20.5

17.2

20.6

18.6

18.1

13.0

18.4

2.2

8.4

8.7

0.9

202.3

95.1

S.D.

7.2

8.5

6.4

5.4

5.5

2.4

3.3

3.0

7.2

5.4

5.0

3.2

3.2

40.8

16.2

N

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

 

Low Concn

(1000 ppm)

Mean

34.6

27.3

30.0**

20.3

14.4

21.1

13.8*

11.8

13.3*

7.9**

7.1

11.2

-0.4

212.4

95.1

S.D.

5.1

5.5

5.9

4.0

6.3

5.3

7.3

8.3

6.3

5.5

4.1

2.6

3.2

28.6

11.1

N

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

 

Intermediate

Concn

(3000 ppm)

Mean

28.7

20.6

20.2

17.6

17.2

15.9

12.8*

13.3

5.8**

10.9**

6.2

7.6

1.0

177.8

82.8

S.D.

6.8

6.3

6.2

4.9

4.2

5.7

3.3

2.7

3.5

2.8

2.9

2.1

4.3

22.8

10.4

N

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

 

High Concn

(7500 ppm)

Mean

27.8

25.5

18.7

17.6

17.4

14.5

9.7**

18.3

9.8**

8.6**

7.6

6.7

-0.6

181.6

83.5

S.D.

5.1

4.8

5.7

5.9

7.4

4.8

5.0

6.1

7.9

4.0

3.3

4.4

4.1

21.3

8.4

N

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

*  Significantly different from control group p<0.05

**  Significantly different from control group p<0.01

Table 9. 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

16.9

10.3

8.4

9.3

7.6

6.1

4.8

6.4

2.8

0.9

7.0

1.1

3.5

85.1

51.2

S.D.

5.0

6.9

4.7

5.0

3.8

3.1

6.9

8.2

6.6

5.5

9.5

5.1

9.6

13.8

7.5

N

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

 

Low Concn

(1000 ppm)

Mean

11.3**

10.2

10.9

9.6

4.7

4.5

4.5

5.2

2.4

2.8

2.8

4.7

6.3

79.9

46.9

S.D.

4.1

5.0

3.9

3.7

2.2

4.0

4.0

2.5

2.6

5.6

6.0

6.1

5.7

16.9

8.9

N

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

 

Intermediate

Concn

(3000 ppm)

Mean

12.2**

9.7

10.3

8.3

8.4

3.6

5.0

4.5

4.3

4.0

-0.6

2.9

4.7

77.3

47.5

S.D.

3.9

6.9

4.8

4.4

3.3

3.5

4.0

3.7

2.9

3.5

3.2

3.9

4.2

20.6

11.2

N

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

 

High Concn

(7500 ppm)

Mean

10.1**

6.0

5.6

9.1

7.0

0.7**

3.6

3.3

5.0

4.0

0.2

0.3

4.5

59.4**

36.5

S.D.

2.5

4.4

3.7

2.9

2.7

3.3

3.3

4.2

3.8

2.9

3.4

3.2

4.2

8.0

5.0

N

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

*  Significantly different from control group p<0.05

**  Significantly different from control group p<0.01

Applicant's summary and conclusion

Conclusions:
Based on the effects on body weight, body weight gain and, to a lesser extent, food consumption, observed among females exposed to diets containing 7500 ppm of the test item, the No Observed Adverse Effect Level (NOAEL) for systemic toxicity of Rosin, reaction products with formaldehyde (CAS 91081-53-7) in this Ninety Day Toxicity study was considered to be 3000 ppm of the test item (equivalent to mean achieved dosages of approximately 213.1 mg/kg bw/day or 255.2 mg/kg bw/day for males and females, respectively). As an initial transient effect on body weight and food consumption (which may reflect palatability of the diet formulations) was apparent at 1000 ppm, the No Observed Effect Level (NOEL) for systemic effects could not be established.
Executive summary:

In a key oral repeated dose toxicity study, the test item (Rosin, reaction products with formaldehyde; CAS# 91081-53-7), was administered by continuous dietary admixture to three groups, each of ten male and ten female Wistar Han™:RccHan™:WIST strain rats, for ninety consecutive days, at dietary concentrations of 1000, 3000 and 7500 ppm (equivalent to a mean achieved dosage of 68.9, 213.1 and 509.2 mg/kg bw/day for males and 84.0, 255.2 and 644.6 mg/kg bw/day for females respectively). A control group of ten males and ten females were treated with 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 for the high dietary level and control animals was performed. Histopathology examinations were also extended to cover the liver and thyroid from low and intermediate dietary level male animals.

 

No mortality was observed through the study period and there were no obvious effects of dietary exposure at 1000, 3000 or 7500 ppm of the test material. Water consumption, behaviour, functional performance and sensory reactivity remained unaffected post dietary exposure at 1000, 3000 or 7500 ppm of the test material. Ophthalmoscopic examinations of animals exposed to 7500 ppm of the test item did not indicate any effect of treatment. Gross necropsy did not reveal any remarkable effects and biochemistry investigations did not reveal any effects of dietary exposure at concentrations of 1000, 3000 or 7500 ppm of the test material.

 

Dietary exposure to the test material at 7500 ppm was associated with statistically significant (p<0.01) lower mean body weight gain compared with control for females during the first week of the study. This lower body weight gain resulted in a statistically significant (p<0.05) lower mean body weight at the end of this first week (Day 8) and statistically significant lower body weights, compared to control, persisted until study termination. Overall body weight gain from the start of treatment to termination was also statistically significantly (p<0.01) lower than control, although lower weekly body weight gains were statistically significant only during Week 6 (Days 36-43). Food consumption for these females was generally lower than control throughout the study, with differences being most noticeable during the first two weeks (Days 1-15), Week 11 (Days 71-78) and final week (Days 85-91) of the study. Females in the 7500 ppm group also showed lower food conversion efficiency compared to control during Weeks 1 (Days 1-8), 6 (Days 36-43) and 11 (Days 71-78) of the study.

 

Females exposed to 1000 or 3000 ppm of the test material showed statistically significant lower mean body weight gain compared with control during the first week of dietary exposure. However, these group mean values showed no relationship to dietary exposure. Food consumption was also lower than control during this period, with mean intake at both dietary levels falling below that observed for animals receiving 7500 ppm. Subsequent body weight gain and food consumption for the remainder of the study in the 1000 or 3000 ppm groups were generally similar to control, with no statistically significant differences from control being observed for mean body weights, weekly body weight gain or overall body weight gain. The initial effects observed for body weight, body weight gain and food consumption may reflect an initial reluctance of the females to eat the diet due to palatability. In view of their transient nature and at the level observed, these findings were considered insufficient to represent an adverse effect of test item exposure. Intergroup differences in body weight gain, food consumption and food conversion efficiency did not indicate any consistent effect of treatment for males exposed to 1000, 3000 or 7500 ppm of the test item.

 

Males exposed to 1000, 3000 or 7500 ppm of the test material showed statistically significant lower body weight gains during Week 7 and Week 9 of the study and statistically significant higher body weight gains during Week 10 compared to control. In view of the absence of any statistically significant differences from control for mean overall body weight gain or mean body weights, these isolated differences were considered to be of no toxicological

significance and insufficient to indicate an adverse effect of test item exposure. Additionally for males exposed to 1000 ppm of the test item, statistically significant higher body weight gain compared to control were apparent during Week 4, but in the absence of any similar effects at higher exposure levels, this isolated finding was considered incidental and unrelated to treatment.

 

Males exposed to 7500 ppm of the test material showed statistically significant, lower haemoglobin and haematocrit compared with control; for haemoglobin, the group mean value was also below the historical control range. No similar statistically significant differences from control for haemoglobin or haematocrit were apparent among females at this exposure level or for males at lower exposure levels. The reduced mean haemoglobin and haematocrit values for males at 7500 ppm of the test item occurred in the absence of any statistically significant differences from control in mean cell haemoglobin concentration, mean cell volume or bilirubin. There were no supporting histopathological findings apparent in the spleen or bone marrow indicative of alterations in erythrocyte production or storage. Consequently, in view of the lack of associated histopathological change and at the level observed, this haematology finding was not considered to represent an adverse effect of exposure to 7500 ppm of the test material.

 

Males exposed to 7500 ppm of the test material showed statistically significant higher numbers of platelets compared with control, with the group mean value being above the historical control range. This change in platelet number occurred in the absence of any histopathological changes in the bone marrow, and was therefore not considered to represent an adverse effect of exposure to 7500 ppm of the test material.

 

Males exposed to 7500 ppm of the test material also showed statistically significant longer prothrombin time compared to control, but there was no similar statistically significant difference in activated partial thromboplastin time (APTT). The group mean prothrombin time for these exposed animals was within the historical control range, while the group mean control value was outside of the historical control range. While higher platelet count may have influenced clotting time, an association with treatment appears unlikely and the statistical significance from control observed for prothrombin time was considered to reflect an abnormally low control value, rather than an indication of an effect of exposure to the test material.

 

Females exposed to 1000, 3000 or 7500 ppm of the test material showed statistically significant lower total leukocyte count compared to control, principally due to a statistically significant lower number of lymphocytes. However, group mean values showed no relationship to exposure and were all within the respective historical control range, while the control value for leukocyte count exceeded the historical control range. An association with treatment for this isolated finding appears unlikely and the statistically significant differences from control were considered to reflect the low control values rather than an indication of an effect of exposure to the test material.

 

Males exposed to 1000, 3000 or 7500 ppm of the test material showed statistically significant higher albumin/globulin ratio compared to control, although there was no consistent relationship to exposure levels. There was no accompanying statistically significant differences from control apparent for total protein or albumin level and group mean values were all within the historical control range. There was no similar increase in albumin/globulin ratio apparent for treated females. In view of its isolated nature and the lack of relationship to exposure, this finding was considered incidental and unrelated to exposure to the test material.

 

Necropsy findings were unremarkable, however, males exposed to 3000 and 7500 ppm of the test item showed statistically significantly higher absolute and relative liver weight compared to control. Supporting this, histopathology examination revealed centrilobular hepatocyte hypertrophy in 4/10 males exposed to 7500 ppm of the test item. These findings were considered to be suggestive of an adaptive (non-adverse) response to mixed function oxidase induction in the liver (Cattley et al., 2002), although this did not result in any obvious disturbance for blood chemistry parameters for either sex. There was no evidence of similar adaptive change for females on the study, although this may be occurring at a lower level, and this apparent difference between the sexes may explain why females appeared to be showing a greater response in terms of body weight gain/food consumption to dietary exposure to the test material than males within this study. Histopathology examinations also revealed the presence of follicular cell hypertrophy in 1/10 and 4/10 males exposed to 3000 and 7500 ppm of the test item, respectively. At the high exposure level, this finding was considered to represent increased hepatic clearance of thyroid hormones causing hypertrophy of follicular cells (Capen et al., 2002, Zabka et al., 2011), therefore the findings in the liver and thyroid gland are considered to be linked. The occurrence of thyroid follicular hypertrophy in one 3000 ppm group male is considered to be a chance finding due to natural variation and not related to exposure to the test material.

 

Based on the effects on body weight, body weight gain and, to a lesser extent, food consumption, observed among females exposed to diets containing 7500 ppm of the test material, the No Observed Adverse Effect Level (NOAEL) for systemic toxicity of Rosin, reaction products with formaldehyde (CAS 91081-53-7) in this Ninety Day Toxicity study was considered to be 3000 ppm (equivalent to mean achieved dosages of approximately 213.1 mg/kg bw/day or 255.2 mg/kg bw/day for males and females, respectively). As an initial transient effect on body weight and food consumption (which may reflect palatability of the diet formulations) was apparent at 1000 ppm, the No Observed Effect Level (NOEL) for systemic effects could not be established.