Registration Dossier

Administrative data

Description of key information

Oral toxicity: NOAEL 2000 ppm, equivalent to 158.15 mg/kg bw/day in males and 183.89 mg/kg bw/day in females, study performed to generally accepted scientific principles with a sufficient level of reporting, Lankas (1981).

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
27 January 1981 - 28 April 1981
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study conducted in accordance with generally accepted scientific principles in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.
Reference:
Composition 0
Composition 0
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
GLP compliance:
no
Remarks:
The study was subjected to auditing procedures
Limit test:
no
Test material information:
Composition 1
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Strain: CD (Sprague-Dawley derived).
- Age at study initiation: Approximately 6 weeks.
- Housing: Individually in elevated stainless steel cages.
- Diet: Standard laboratory diet ad libitum. Fresh food was presented once weekly.
- Water: ad libitum. By automated water system.
- Acclimation period: 14 Days (13 - 26 January 1981).

ENVIRONMENTAL CONDITIONS
- Photoperiod (hrs dark / hrs light): 12 hour light/dark cycle.

IN-LIFE DATES: From: To: 27 January 1981 - 28 April 1981
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
Appropriate amounts of test substance were mixed with standard laboratory diet weekly.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of each dietary admixture for each dose level were taken in duplicate, one sample for each dose level was analyzed for concentration of test material; the other was stored frozen for the first four weeks of the study. After four weeks, samples were analysed once every 2 weeks and the samples for off weeks were stored frozen.
Duration of treatment / exposure:
Males: 90 days
Females: 91 days
Frequency of treatment:
Daily: the test material was administered in the animals' feed, available ad libitum. Fresh food was presented once weekly.
Remarks:
Doses / Concentrations:
0, 2000, 10000 and 50000 ppm
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
0.0, 158.15, 775.74, 3865.67 mg/kg bw/day males
Basis:
actual ingested
Remarks:
Doses / Concentrations:
0.0, 183.89, 933.09, 4633.36 mg/kg bw/day females
Basis:
actual ingested
No. of animals per sex per dose:
- 2000 and 10000 ppm dose level: 20 animals per sex per dose.
- 0 and 50000 ppm dose level: 30 animals per sex per dose.
Control animals:
yes, plain diet
Details on study design:
The additional 10 animals in the 0 and 50000 ppm dose groups were held for a 1 month recovery period during which no test material was administered. The purpose of the recovery period was to assess the reversibility of any toxic effects noted.
Observations and examinations performed and frequency:
OBSERVATIONS
- Mortality and gross signs of toxicologic or pharmacologic effects: twice daily.
- Detailed physical examination for signs of local or systemic toxicity, pharmacologic effects and palpation for tissue masses: weekly.
- Bodyweight: twice pre-test, weekly during treatment and terminally (after fasting).
- Food consumption: weekly, beginning one week prior to treatment.
- Test substance intake: calculated from weekly body weight and food consumption data. Based on nominal concentrations.

LABORATORY STUDIES
Blood was obtained via venipuncture of the orbital sinus (retrobulbar venous plexus) under light ether anaesthesia. Animals were selected randomly; the same animals were used at all intervals when feasible. Rats were fasted overnight prior to blood collections and were not dosed until after samples were collected.
10 males and 10 females were examined pre-test; 10 animals per sex per group were examined at 7 and 13 weeks.

PARAMETER EVALUATED
-Haematology
Haemoglobin haematocrit
Erythrocytes platelets
total leukocytes differential leukocytes

-Clinical Chemistry
serum glutamic oxaloacetic transaminase serum glutamic pyruvic transaminase
alkaline phosphatase blood urea nitrogen
fasting glucose total protein
cholesterol sodium
potassium chloride
calcium creatinine
total bilirubin direct bilirubin
* Due to a technical error Albumin was not evaluated for pre-test blood work, therefore Globulin and A/G Ratio could not be calculated.

-Urinalysis
gross appearance specific gravity
pH protein
glucose ketones
bilirubin occult blood
urobilinogen
Sacrifice and pathology:
POSTMORTEM
- Animals dying spontaneously or killed in a moribund condition: subject to complete gross postmortem examination.

NECROPSY
- All animals were subjected to necropsy.
- Sacrifice method: exsanguination under ether anaesthesia.
- Organs weighed and organ/bodyweight ratios calculated: (paired organs weighed separately)
kidneys ovaries
liver spleen
heart pituitary
brain adrenals
testes thyroid/parathyroid

TISSUES PRESERVED (numbers in parentheses indicate number of organs/sections preserved).
adrenal (2) aorta (abdominal)
bone (sternum, femur) bone marrow (sternum)
brain cecum
colon duodenum
epididymis (2) eye (2-with optic nerve)
heart ileum
jejunum kidney (2)
liver lungs (with mainstem bronchi)
lymph node (mesenteric) mammary gland (inguinal)
nerve (sciatic) oesophagus
ovaries pancreas
pituitary prostate/seminal vesicles
rectum salivary gland (submandibular)
skeletal muscle (right bicep femoris) skin (with mammary gland)
spinal cord (cervical) spleen
stomach testes
thymus thyroid/parathyroid
trachea urinary bladder
uterus
gross lesions (including a section of normal-appearing portion of same tissue)
tissue masses
entire head

- Preservatives: 10 % neutral buffered formalin used for all tissues.
- Stains: Haematoxylin and Eosin
- Tissues examined histopathologically: slides of all tissues listed were prepared and examined microscopically for control and high dose animals. In addition, the livers and kidneys from all dosed, control and recovery animals were examined microscopically. Livers from six randomly selected high dose animals were stained with Oil Red-O for lipids.
- As an addendum to the pathology report, microscopic examination of the thyroid glands from the 2000 and 10000 ppm dose groups, male and female, was performed.
Statistics:
If a standard error for one treated group is 0.0, or when N (number of animals) is less than or equal to two animals for any treated group, the variances of the two groups remaining were tested for equality using the F-test. If the N (number of animals) for the control group is less than or equal to two animals, no statistics are presented due to lack of variance.

Statistical evaluation of equality of means was made by the appropriate one way analysis of variance technique, followed by a multiple comparison procedure if needed. First, Bartlett's test was performed to determine if groups had equal variance. If the variances were equal, parametric procedures were used; if not, nonparametric procedures were used. The parametric procedures were the standard one way ANOVA using the F distribution to assess significance. If significant differences among the means were indicated, Dunnett's test was used to determine which means were significantly different from the control. If a nonparametric procedure for testing equality of means was needed, the Kruskal-Wallis test was used, and if differences were indicated a summed rank test (Dunn) was used to determine which treatments differed from control.

A statistical test for trend in the dose levels was also performed. In the parametric case (i.e. equal variance) standard regression techniques with a test for trend and lack of fit were used. In the nonparametric case Jonckheere's test for monotonic trend was used.
The test for equal variance (Bartlett's) was conducted at the 1 %, two-sided risk level. All other statistical tests were conducted at the 5 % and 1 %, two-sided risk level.

The variances of the two groups were tested for equality using the F test. If the variances were equal, a standard independent two sample t-test was used to determine equality of means. If the variances differed, Welch's t-test was used to determine equality of means. All tests were conducted at the 5 % and 1 %, two-sided risk level.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
See below
Mortality:
mortality observed, treatment-related
Description (incidence):
See below
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
See below
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
See below
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
See below
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
See below
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
See below
Histopathological findings: neoplastic:
no effects observed
Details on results:
MORTALITY
Four high dose males died during the course of this study. Two (Nos. 704 and 725) were found dead after 48 and 63 days on test. Neither animal showed any significant in-life observations which would account for their deaths. A further two animals (Nos. 719 and 727) showed decreased food consumption, red nasal discharge, and cool body temperature. One (No. 719) died spontaneously on test day 90, while the latter was euthanised on test day 63.
In addition, two females (No. 403 from the 2000 and No. 804 from the 50000 ppm dose groups) died following eye bleeding during Week 7. All remaining animals survived the duration of the study and were sacrificed either on test day 91 or on test day 120, following the one month recovery period.

CLINICAL OBSERVATIONS
Physical observations noted during the course of this study included excessive lacrimation, swollen salivary glands and alopecia. These observations occurred sporadically in both sexes of all groups, including the controls, in about 10 - 20 % of all animals. These findings are not considered to be related to the administration of the test substance.
One finding, however, did tend to occur more frequently in the treated animals compared to the controls. Red and/or mucoidal nasal discharge occurred in the high dose males at an incidence of approximately 10 - 20 %, compared to about 3 - 6 % in the control males. This observation occurred mainly during weeks 8 to 12 of the study and was not evident to any significant extent during the recovery period. The incidence of this finding in the 2000 and 10000 ppm dose group males was similar to the control group. Females were not significantly affected.

BODYWEIGHT
Mean bodyweights for both sexes in the 2000 and 10000 ppm dose groups were similar to the control values during the course of this study.
However, the high dose animals of both sexes exhibited a statistically significant reduction in mean bodyweights during the latter phase of the treatment period (p<0.01). At Week 13 there was about a 10 and 7 % reduction in group mean bodyweights (males and females respectively) as compared to their control groups. During the recovery period, these bodyweight effects were still evident in both sexes. However, it was only in the males that this was statistically significant; although this effect decreased as the recovery period progressed.

FOOD CONSUMPTION
Mean food consumption values for the treated groups of both sexes did not differ markedly from their respective control groups. During the latter phase of the treatment period, a slight trend toward increased food consumption occurred in both the treated males and females. This trend continued during the recovery period with consistently elevated values noted in both the high dose males and females, as compared to their respective controls. This trend was probably due to the high content of the test material in the treated diets with the resultant decrease in body weights in the animals, as noted above. This would tend to increase food consumption on a (g/kg/day) basis.

- Test Substance Intake
Since food consumption values were similar among the treated groups and controls for both sexes, test substance intake values for the treated groups were proportional to the nominal dietary concentrations of the test substance. As expected, during the first several weeks of the study the animals consumed more feed on a body weight basis and, therefore, test substance intake values were also higher, as compared to the latter weeks of the study.

HAEMATOLOGY
Pre-test values for both sexes were within normal limits for rats of this age and strain. At weeks 7 and 13 there was a trend toward slightly reduced haemoglobin levels in both sexes of the high dose group. However, these values were within the normal physiological range for this species and strain, as were all other haematology parameters evaluated.

CLINICAL CHEMISTRY
Pre-test clinical chemistry parameters were within the normal physiological range for rats of this age and strain. Relatively high alkaline phosphatase values were seen but these are expected in immature animals.
At week 7, there was a statistically significant increase in the SGPT level in the high dose males (p<0.01), while in the high dose females, alkaline phosphatase and cholesterol were increased (p<0.05).
At week 13 a trend toward increased BUN values was evident in the 10000 and 50000 ppm dose level males. Bilirubin values (both direct and total) were significantly lower in the high dose animals of both sexes at weeks 7 and 13.
Other statistically significant values were evident at both the 7 and 13 week intervals. However, these are considered to be biologically insignificant as the means of these values fall within the normal physiological range for the laboratory rat.
The effects on SGPT and BUN levels in the high dose males are suggestive of a toxic effect in the liver and kidney. The effects on cholesterol and alkaline phosphatase in the high dose females also suggest the liver as a target organ. The data indicate a minimal degree of damage in these organs in the high dose animals, as the relative changes in the above parameters are slight.

URINALYSIS
All urinalysis parameters measured were within normal limits at both the 7 and 13 week evaluations for all groups, both males and females.

ORGAN WEIGHTS AND ORGAN/BODY WEIGHT RATIOS
Among the males, a statistically significant decrease in the mean absolute brain weight occurred in the 50000 ppm dose group, while the spleen/body weight ratio was significantly increased (p<0.05). A trend toward increased absolute organ weight as well as organ/body weight ratios was evident for the kidneys, thyroid and liver in the treated males. These increases were statistically significant in the high dose males for the kidneys and liver and in all treatment groups for the thyroids (p<0.01).
Among the females, the brain and spleen organ/body weight ratios were increased in all treatment groups. These effects were statistically significant for the brain/body and spleen/body weight ratios in the 50000 ppm dose group. In addition, a trend toward increased ovary/body weight ratios was found for all the treatment groups. A trend toward increased absolute organ weight and organ/body weight ratios was evident in the females for the kidneys and liver in dose levels 10000 and 50000 ppm and for the thyroids in all treatment groups.
For the most part, the above increases in relative organ/body weight ratios are probably due to the reduced terminal body weights for the treated animals without a concomitant decrease in the absolute organ weight. However, for the livers and kidneys in both sexes, these parameters are fairly sensitive indicators of damage for both of these organs. Therefore, this data confirms the clinical chemistry data which indicated a slight toxic effect in the livers and kidneys in the high dose animals. The other organ weight changes noted above were not reflected in any unusual gross or microscopic changes.
At the end of the recovery period, the increase in the kidney/body weight ratio was still evident in the high dose males, while the increases in the liver and thyroid organ/body weight ratios were no longer present. Among the high dose females, all of the organ weight effects previously noted were no longer present.

PATHOLOGY
Gross postmortem findings in the animals of this study occurred sporadically in both sexes of the treated and/or control animals. They did not appear to be related to the administration of the test substance.

Microscopic evaluations of tissues from the terminal sacrifice animals revealed a significant incidence of hepatic cytoplasmic vacuolisation in the high dose group. Sections stained with Oil Red-O confirmed the presence of lipid in these vacuoles. The males were more severely affected than the females. After one month of recovery, this effect was still evident in the males, although to a lesser degree. Female rats were almost completely recovered. Minimal fatty change was evident in the 10000 ppm dose group, while the 2000 ppm dose group was unaffected.
Microscopic observation of the kidneys from high dose males indicated a minimal to moderate necrosis of tubular epithelial cells with protein accumulation in the lumen. After one month of recovery, 4 of 10 animals were still affected. In the high dose females, only 2 of 20 animals were affected. None of the female recovery animals showed evidence of renal damage. Animals from dose groups 2000 and 10000 ppm did not exhibit renal lesions.
Microscopic examination of the thyroid glands from all groups showed that there was no significant difference in the microscopic appearance of the thyroid glands between the treated and control groups of rats.
Other microscopic findings occurred sporadically in the treated and/or control groups and therefore, did not appear to be related to the administration of the test substance.
Dose descriptor:
NOEL
Effect level:
2 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: 2000 ppm is equivalent to 158.15 mg/kg bodyweight/day for males and approximately 183.89 mg/kg bodyweight/day for females.
Critical effects observed:
not specified

Key to Abbreviations and Symbols

SD = Standard Deviation                                                   N = Number of animals

SGPT = Serum Glutamic Pyruvic Transaminase                       ALKPHOS = Alkaline Phosphatase

BUN = Blood Urea Nitrogen                                              CHOL = Cholesterol

TBILI = Total Bilirubin                                                    DBILI = Direct Bilirubin

*Significantly different from the control at p≤0.05

**Significantly different from the control at p≤0.01

 

Table 2 Male Clinical Chemistry Summary Data

Time Period

Dose Level (ppm)

 

SGPT

(IU/L)

 

ALKPHOS

(IU/L)

 

BUN

(mg/dL)

 

CHOL

(mg/dL)

 

TBILI

(mg/dL)

 

DBILI

(mg/dL)

 

Pre-test

0

Mean

SD

N

25.0

4.0

9

386.0

96.0

9

17.7

1.2

9

75.5

13.1

7

0.08

0.03

9

0.06

0.02

3

 

 

 

 

Week 7

0

Mean

SD

N

17.0

1.0

1

123.0

29.0

10

16.5

2.1

10

57.0

8.0

10

0.04

0.03

10

0.03

0.03

10

2000

Mean

SD

N

17.0

2.0

10

124.0

28.0

10

15.7

2.0

10

43.0*

11.0

10

0.02

0.01

10

0.02

0.01

10

10000

Mean

SD

N

16.0

3.0

10

129.0

28.0

10

16.6

2.4

10

55.0

8.0

10

0.06

0.03

10

0.03

0.02

10

50000

Mean

SD

N

23.0**

12.0

10

118.0

29.0

10

17.9

2.5

10

59.0

16.0

10

0.03

0.01

10

0.01

0.03

10

 

 

 

 

Week 13

0

Mean

SD

N

19.0

2.0

10

81.0

19.0

10

15.9

1.4

10

57.0

10.0

10

0.08

0.03

9

0.05

0.03

9

2000

Mean

SD

N

19.0

5.0

10

82.0

15.0

10

15.4

1.5

10

49.0

18.0

10

0.06

0.03

10

0.03

0.02

10

10000

Mean

SD

N

16.0

4.0

10

87.0

17.0

10

17.4

1.3

10

63.0

11.0

10

0.08

0.03

9

0.09

0.04

9

50000

Mean

SD

N

29.0

13.0

10

82.0

18.0

10

18.1*

2.1

10

81.0

34.0

10

0.04**

0.01

10

0.04

0.01

10

 

Table 3 Female Clinical Chemistry Summary Data

Time Period

Dose level (ppm)

 

SGPT

(IU/L)

 

ALKPHOS

(IU/L)

 

BUN

(mg/dL)

 

CHOL

(mg/dL)

 

TBILI

(mg/dL)

 

DBILI

(mg/dL)

 

Pre-test

0

Mean

SD

N

17.0

2.0

9

319.0

88.0

9

17.1

3.2

9

69.3

22.1

6

0.11

0.03

9

0.02

0.01

4

 

 

 

 

Week 7

0

Mean

SD

N

23.0

4.0

9

54.0

13.0

9

18.8

2.6

9

68.0

16.0

9

0.06

0.10

8

0.05

0.03

8

2000

Mean

SD

N

22.0

4.0

9

66.0

11.0

9

20.3

5.9

9

58.0

13.0

9

0.03

0.01

9

0.02*

0.02

9

10000

Mean

SD

N

18.0

4.0

9

57.0

11.0

9

16.8

2.0

9

70.0

22.0

9

0.08

0.02

9

0.05

0.04

9

50000

Mean

SD

N

22.0

 8.0

9

78.0*

26.0

9

18.0

1.8

9

91.0*

18.0

9

0.05

0.04

9

0.02*

0.01

9

 

 

 

 

Week 13

0

Mean

SD

N

23.0

13.0

10

39.0

9.0

10

17.1

2.4

10

98.0

23.0

10

0.31

0.02

10

0.10

0.04

10

2000

Mean

SD

N

24.0

12.0

10

49.0

12.0

10

17.4

2.3

10

87.0

17.0

10

0.08*

0.02

10

0.06

0.01

10

10000

Mean

SD

N

21.0

6.0

10

37.0

9.0

10

16.6

1.6

10

77.0

17.0

10

0.08**

0.01

10

0.08

0.03

10

50000

Mean

SD

N

18.0

9.0

10

47.0

13.0

10

18.4

2.2

10

100.0

27.0

10

0.04**

0.02

10

0.04**

0.01

10

 

Table 4 Male Summary Data for Organ Weights and Organ/Bodyweight Ratios

Dose level (ppm)

 

Terminal Bodyweight

(g)

Right Kidney

Left Kidney

Liver

Thyroid

Weight (g)

Org/TBW

(x 1000)

Weight (g)

Org/TBW

(x 1000)

Weight (g)

Org/TBW

(x 100)

Weight (g)

Org/TBW

(x 100000)

0

Mean

SD

N

427.0

33.0

20

1.370

0.157

20

3.21

0.24

20

1.428

0.305

20

3.36

0.73

20

10.879

1.253

20

2.55

0.16

20

0.0129

0.0042

18

3.00

0.92

18

2000

Mean

SD

N

427.0

30.0

20

1.436

0.133

20

3.36

0.22

20

1.414

0.150

20

3.31

0.24

20

11.669

1.751

20

2.72

0.27

20

0.0202**

0.0028

20

4.75**

0.73

20

10000

Mean

SD

N

422.0

30.0

20

1.462

0.143

20

3.47*

0.35

20

1.425

0.129

20

3.39

0.33

20

11.869

1.155

20

2.81**

0.11

20

0.0238**

0.0030

20

5.67**

0.87

20

50000

Mean

SD

N

399.0*

33.0

16

1.571**

0.155

16

3.95**

0.33

16

1.516

0.164

16

3.81**

0.30

16

16.453**

2.098

16

4.12**

0.35

16

0.0176**

0.0056

15

4.41**

1.32

15

 

Table 5 Female Summary Data for Organ Weights and Organ/Bodyweight Ratios

Dose level (ppm)

 

Terminal Bodyweight

(g)

Right Kidney

Left Kidney

Liver

Thyroid

Weight (g)

Org/TBW

(x 1000)

Weight (g)

Org/TBW

(x 1000)

Weight (g)

Org/TBW

(x 100)

Weight (g)

Org/TBW

(x 100000)

0

Mean

SD

N

243.0

16.0

20

0.836

0.077

20

3.45

0.31

20

0.820

0.076

20

3.38

0.26

20

6.557

0.559

20

2.70

0.17

20

0.0108

0.0035

18

4.51

1.51

18

2000

Mean

SD

N

233.0

20.0

19

0.839

0.070

19

3.61

0.24

19

0.827

0.055

19

3.56

0.20

19

6.492

0.604

19

2.79

0.18

19

0.0139*

0.0021

18

5.98**

0.78

18

10000

Mean

SD

N

236.0

19.0

20

0.854

0.089

20

3.62

0.33

20

0.830

0.077

20

3.53

0.34

20

7.247**

0.654

20

3.07**

0.21

20

0.0180**

0.0032

18

7.71**

1.55

18

50000

Mean

SD

N

223.0*

19.0

19

0.876

0.097

19

3.92**

0.24

19

0.860

0.103

19

3.85**

0.29

19

9.111**

0.955

19

4.08**

0.33

19

0.0145**

0.0033

18

6.52**

1.46

18

Conclusions:
The data indicate toxic effects in the liver and kidneys due to the administration of the test substance in the 50000 ppm dose group males and females.
These effects appear to be reversible based on microscopic evaluations of these organs in the recovery animals. The evidence is equivocal with regard to toxicity in the 10000 ppm dose group, while the 2000 ppm dose appears to be the NOEL. This nominal dose level is equivalent to an actual ingested dose level of 158.15 mg/kg bw/day in males and 183.89 mg/kg bw/day in females over the 13 week exposure period. Since the repeated dose toxicity of the test substance is >100 mg/kg day/bw classification is not required.
Executive summary:

A 90-day study was conducted to assess the toxicity of the test material broadly in accordance with OECD guideline 408.

 

The test material was administered via dietary admixture, 7 days per week to CD (Sprague-Dawley derived) rats for three months. Dose levels were 0, 2000, 10000 and 50000 ppm. Rats were randomly divided into four groups of 30 rats/sex/group in the 0 and 50000 ppm dose groups and 20 rats/sex/group in the 2000 and 10000 ppm dose groups.

All animals were subject to necropsy at the termination of the study. This was at 90 days for 20 males and 91 days for 20 females from all groups; the remaining 10 animals were subjected to a one month recovery period and necropsied when this was completed.

 

- Two females and four males died during the course of the study. A significant physical observation noted was red/mucoidal nasal discharge. This occurred more frequently in the high dose males during the latter phase of the treatment period. During the recovery phase, this observation was no longer evident.

- A significant bodyweight decrease was observed during the latter portion of the study in the high dose animals of both sexes. This decrease persisted throughout the recovery period.

- Clinical chemistry effects were also noted. At Week 7, serum glutamic pyruvic transaminase was elevated in the high dose males, while alkaline phosphatase and cholesterol were elevated in the high dose females. At Week 13, blood urea nitrogen values were increased in the 10000 and 50000 ppm dose level males. Direct and total bilirubin values were reduced in the high dose animals at both 7 and 13 weeks.

- At the 13 week necropsy, a trend toward increased absolute and relative (to body weight) organ weights was evident for the kidneys and liver for the males in the 10000 and 50000 ppm dose groups and for the thyroids in all treatment groups. Among the females, a trend toward increased absolute and relative organ weights were found for the liver, thyroids and kidneys in all treatment groups.

- Microscopic evaluation of selected tissues indicated slight cellular necrosis in the proximal tubules of the kidney and lipid deposition in livers of the high dose animals only. The males were more sensitive to these effects than the females. These findings were still evident in the recovery animals, although at a lesser incidence and degree.

 

The data indicate toxic effects in the liver and kidneys due to the administration of the test substance in the 50000 ppm dose group males and females.

These effects appear to be reversible based on microscopic evaluations of these organs in the recovery animals. The evidence is equivocal with regard to toxicity in the 10000 ppm dose group, while the 2000 ppm dose appears to be the NOEL. This nominal dose level is equivalent to an actual ingested dose level of 158.15 mg/kg bw/day in males and 183.89 mg/kg bw/day in females over the 13 week exposure period. The repeated dose toxicity of the test substance is therefore above the limits of classification.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
158.15 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
In addition to the key study two additional studies performed on the registered substance have been provided as supporting data. The first is a subchronic 90 day study, the second a subacute 28 day study. Furthermore, six studies performed on a structural analogue (4,4’-thiobis(6-t-butyl-m-cresol)) have been provided on the basis of read-across. All studies support the findings of the key study and have been discussed in detail below. The overall quality of the dataset is considered to be high and sufficient for addressing the repeat dose toxicity endpoint.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Mode of Action Analysis / Human Relevance Framework

Additional information

ORAL TOXICITY

The repeated dose toxicity via oral exposure has been addressed using a key study performed on the substance to be registered. Two further studies on the substance have been provided to support these findings, in the form of a subacute (28 day) and sa ubchronic (90 day) oral toxicity study. Additional subacute, subchronic and chronic studies have been provided as supporting information, performed on the structural analogue (4,4’-thiobis(6-t-butyl-m-cresol)) and provided on the basis of read-across.

 

2,2'-Methylenebis(6-nonyl-p-cresol) Data

90 Day Rat Study: In the key study Lankas (1981) the oral toxicity of the registered substance was assessed in a 90-day feeding study, which was conducted broadly in accordance with OECD guideline 408. The study was performed to a good standard with a sufficient level of reporting and assigned a reliability score of 1 in accordance with Klimisch (1997). Rats were allowed access to dosed feed at levels of 0, 2000, 10000 and 50000 ppm, ad libitum 7 days per week. Thirty rats/sex/dose were treated in the high dose group and the control; 20 rats/sex/dose were treated at the remaining treatment levels. Twenty rats/sex/dose were terminated and necropsied on day 90/91, the remaining 10 in the control and high dose groups were subjected for a one month recovery period and necropsied when this was completed.

 Under the conditions of the test the following effects were observed:

1) Two females and four males died during the course of the study. A significant physical observation noted was red/mucoidal nasal discharge. This occurred more frequently in the high dose males during the latter phase of the treatment period. During the recovery phase, this observation was no longer evident.

2) A significant bodyweight decrease was observed during the latter portion of the study in the high dose animals of both sexes. This decrease persisted throughout the recovery period.

3) Clinical chemistry effects were also noted. At Week 7, serum glutamic pyruvic transaminase was elevated in the high dose males, while alkaline phosphatase and cholesterol were elevated in the high dose females. At Week 13, blood urea nitrogen values were increased in the 10000 and 50000 ppm dose level males. Direct and total bilirubin values were reduced in the high dose animals at both 7 and 13 weeks.

4) At the 13 week necropsy, a trend toward increased absolute and relative (to body weight) organ weights was evident for the kidneys and liver for the males in the 10000 and 50000 ppm dose groups and for the thyroids in all treatment groups. Among the females, a trend toward increased absolute and relative organ weights were found for the liver, thyroids and kidneys in all treatment groups.

5) Microscopic evaluation of selected tissues indicated slight cellular necrosis in the proximal tubules of the kidney and lipid deposition in livers of the high dose animals only. The males were more sensitive to these effects than the females. These findings were still evident in the recovery animals, although at a lesser incidence and degree.

 The data indicate toxic effects in the liver and kidneys due to the administration of the test substance in the 50000 ppm males and female dose groups.

These effects appear to be reversible based on microscopic evaluations of these organs in the recovery animals. The evidence is equivocal with regard to toxicity in the 10000 ppm dose group, while the 2000 ppm dose appears to be the NOEL. This nominal dose level is equivalent to an actual ingested dose level of 158.15 mg/kg bw/day in males and 183.89 mg/kg bw/day in females over the 13 week exposure period. The repeated dose toxicity of the test substance is therefore beyond the limits of classification.

 

90 Day Rat Study: The supporting study, Anon. (1957), is another subchronic 90 day feeding study. The study pre-dates GLP and standard guidelines. The data are well reported although in limited detail. The experiment was conducted in accordance with generally accepted scientific principles. For a study of this nature it is not possible to accurately assign the reliability based on this test report and was therefore assigned a score of 4 in accordance with Klimisch (1997). During the study the test material was fed to weanling albino rats at levels of 0.2, 1.0 and 5.0 % in a nutritionally adequate diet. The growth and development, the efficiency of food utilisation, haematology and blood chemistry and the gross appearance at autopsy were observed. Even at the 5.0 % level of intake, the ingestion of the test material did not adversely affect the growth, food intake and efficiency of food utilisation. Post-mortem examination revealed no pathology attributable to the test material. Under the conditions of this feeding study, it was concluded that daily ingestion levels of the test material as high as 5.0 % of the diet were essentially innocuous.

 

28 Day Rat Study: The supporting study, Sachsee (1984), is a subacute 28 day feeding study, performed on the registered substance. The study was conducted to GLP in compliance with a standardised guideline (‘Wegleitung der IKS (Interkantonale Kontrollstelle fuer Heilmittel) betreffend gute Laborpraxis fuer nichtklinische Laborversuche’ from April 28, 1980, Berne, Switzerland, amended 1982). The study was reported to a good standard with a sufficient level of reporting to assess the quality of the submitted data. Accordingly the study was assigned a reliability score of 1 in line with Klimisch (1997).

The test material was administered to SPF-bred Wistar rats as a feed admixture at nominal concentrations of 2000, 10000 and 50000 ppm. Under the conditions of the test, no signs of systemic and/or local toxicity were observed and no death occurred. The food consumption and body weight gain of all treated groups were similar to those of the control group.

Significant treatment related pathological results were:

1) The mid- and high-dose males displayed increased liver and kidney weights as well as decreased thyroid weights. The mid- and high-dose females showed increased liver weights only.

2) In the liver, slight to moderate patchy fatty change was encountered in all high-dose males and minimal to slight fatty change was encountered in half of the high-dose females.

The findings indicate that the thyroid gland, the liver and the kidney are considered to be target organs. However, based upon the results of this study, the NOEL of the test material is considered to be 2000 ppm (approximately equivalent to 163 mg/kg bodyweight/day for males and approximately 170 mg/kg bodyweight/day for females). Therefore the oral toxicity of the test material is above the limits of classification.

 

Read-Across Data on 4,4’-thiobis(6-t-butyl-m-cresol)

Several studies performed on a structural analogue have been provided as supporting information. The subacute, subchronic and chronic studies conducted using rats and mice were all reported within the same publication, Alden at al. (1994). The feeding studies were conducted under GLP conditions and performed in accordance with sound scientific principles and followed a guideline similar to either OECD 407 or 408 (28 and 90 days, respectively). The data were assigned a reliability score of 2 in accordance with Klimisch (1997).

 

15 Day Rat Study: Groups of 10 male and 10 female rats were fed diets containing 0, 1000, 2500, 5000, 10 000 or 25 000 ppm of the test material for 15 days. These dose levels were equivalent to ingested doses of 0, 95, 235, 335 and 365 mg/kg bw/day in males and 0, 85, 220, 325 and 270 mg/kg bw/day in females (it was not possible to determine the high dose level due to the observed mortality rate).

All 25 000 ppm rats and three male and four female 10 000 ppm rats died. Surviving rats in the 10 000 ppm groups had a significant weight loss and the final mean body weights of the 5000 and 10 000 ppm groups were significantly lower than those of the controls. Male and female rats exposed to 5000, 10 000 or 25 000 ppm of the test material consumed markedly less feed than the controls, however reduced feed consumption was considered to be a result of feed palatability.

Diarrhoea occurred in 5000, 10 000 and 25 000 ppm males and females. The principal lesions attributed to the administration of the test material were renal papillary and tubule necrosis which occurred in 10 000 ppm rats. Focal necrosis or erosions of the glandular stomach also occurred in some 10 000 ppm rats. Changes were observed in the thymus and spleen in rats receiving 10 000 ppm; however these were attributed to debilitation or stress; depletion in the hematopoietic cells from the bone marrow was observed and this was also attributed to nutrient deficiency accompanying weight loss.

 

13 Week Rat Study: Groups of 10 male and 10 female rats were fed diets containing 0, 250, 500, 1000, 2500 or 5000 ppm of the test material for 13 weeks. Equivalent to doses of 0, 15, 30, 60, 165 and 315 mg/kg bw/day in males and 0, 15, 35, 70, 170 and 325 mg/kg bw/day in females.

All rats survived to the end of the study. The final mean body weight of 5000 ppm males was 40 % lower than that of the controls; the final mean body weight of 5000 ppm females was 27 % lower than that of the controls. Feed consumption by male and female rats exposed to 5000 ppm was markedly lower than that by the controls throughout the study. The absolute and relative liver weights of 5000 ppm females were significantly greater than those of the controls.

Serum alkaline phosphatase levels were significantly higher in 2500 and 5000 ppm males and slightly higher in 5000 ppm females. Serum alanine aminotransferase levels were significantly higher in 2500 and 5000 ppm males and females. Haematocrit and haemoglobin concentrations and mean erythrocyte volume values were significantly lower in 1000, 2500 and 5000 ppm males than in controls; these values were also significantly lower in 5000 ppm females. A dose-related significant increase in forelimb and hindlimb grip strength was observed in exposed male and female rats.

Histopathologic findings in the liver of 2500 and 5000 ppm males and females included hypertrophyof Kupffer cells, bile duct hyperplasia, and individual cell necrosis of hepatocytes; centrilobular hepatocyte hypertrophy also occurred in males and females exposed to 5000 ppm. Macrophages were increased in size and number in the mesenteric lymph nodes of males and females exposed to 5000 ppm, and to a lesser extent in 2500 ppm male and female rats. Pigmentation and degeneration of the renal cortical tubule epithelial cells was also present in males and females in the 2500 and 5000 ppm groups; cortical tubule necrosis occurred in 5000 ppm males and females.

Under the conditions of this study, the NOAEL was 500 ppm based on haematological effects, equivalent to 30 mg/kg bw/day in males and 35 mg/kg bw/day in females.

 

2 Year Rat Study: Groups of 115 male and 75 female rats were fed diets containing 0, 500, 1000 or 2500 ppm of the test material for 104 weeks. Equivalent to ingested dose level of 0, 20, 40, 100 mg/kg bw/day in males and 0, 20, 45 an d 120 mg/kg bw/day in females.

Two year survival rates and mean body weights of exposed male and female rats were generally similar to those of the controls.

A 15 month interim evaluation was conducted, at which the absolute and relative liver weights of 2500 ppm female rats were significantly greater than those of controls; at 15 months and at the end of the study, the incidences of Kupffer cell hypertrophy, hepatocyte cytoplasmic vacuolisation and mixed cell foci were also significantly increased. At the end of the study, the incidence of hepatocellular fatty change was significantly increased in 2500 ppm females. The incidence of Kupffer cell hypertrophy was significantly increased in 2500 ppm males at 15 months and at 2 years; the incidence of cytoplasmic vacuolisation was significantly increased in all exposed males at 15 months but only moderately increased in 1000 and 2500 ppm males at 2 years; the incidence of basophilic foci was significantly increased in 2500 ppm males at 15 months and the incidence of mixed cell foci was significantly increased in 1000 and 2500 ppm male rats at 2 years. The incidences of hepatocellular adenoma or carcinoma (combined) in exposed male rats were not significantly greater than that in the controls. The severity of nephropathy was significantly increased in 2500 ppm female rats.

There was no evidence of carcinogenic activity of the test material in male or female F344/N rats administered 500, 1000 or 2500 ppm of the test material. Non-neoplastic lesions associated with exposure to the test material included: Kupffer cell hypertrophy, cytoplasmic vacuolisation and mixed cell foci in the liver of male and female rats, fatty change in the liver of female rats and an increase in the severity of nephropathy in the kidney of female rats. In addition, decreased incidences of fibroadenoma, adenoma, or carcinoma (combined) were observed in the mammary gland of female rats.

Under the conditions of this study, the NOAEL was 500 ppm (equivalent to 20 mg/kg bw/day) based on the enhancement of seemingly age-related effects in the liver at 1000 ppm. There was no evidence of carcinogenic activity.

 

15 Day Mouse Study: Groups of 10 male and 10 female mice were fed diets containing 0, 1000, 2500, 5000, 10 000 or 25 000 ppm of the test material for 15 days. These dose levels were equivalent to ingested doses of 0, 285, 585 and 475 mg/kg bw/day in males and 0, 360, 950 and 1030 mg/kg bw/day in females (it was not possible to determine the 10 000 and 25 000 ppm dose levels due to the observed mortality rate).

All 10 000 and 25 000 ppm mice died, as did eight males and eight females given 5000 ppm. A significant weight loss occurred in surviving 5000 ppm males and females and the final mean body weights of 2500 ppm females and 5000 ppm males and females were significantly lower than those of the controls. Feed consumption by mice given 5000, 10 000 or 25 000 ppm was markedly reduced; however this reduction was attributed to poor feed palatability. Diarrhoea occurred in all 25 000 ppm mice and in most male and female mice given 5000 or 10 000 ppm. Renal tubule necrosis occurred in eight males and three females in the 5000 ppm groups.

Depletion of cells from the bone marrow and lymphoid organs was observed in many mice in the 5000 ppm group. Bone marrow depletion was attributed to nutrient deficiency accompanying weight loss; depletion of lymphoid organs is commonly associated with low body weight, debilitation, and stress.

 

13 Week Mouse Study: Groups of 10 male and 10 female mice were fed diets containing 0, 100, 250, 500, 1000 or 2500 ppm of the test material for 13 weeks. Equivalent to ingested dose levels of 0, 15, 30, 65, 145 and 345 mg/kg bw/day in males and 0, 10, 35, 60, 165 and 340 mg/kg bw/day in females.

All mice survived to the end of the study. The final mean body weights of 2500 ppm males and of 500, 1000, and 2500 ppm females were significantly lower than those of the controls. Feed consumption by 2500 ppm males averaged 24 % lower than that by controls through week 3 and was similar to that by controls for the remainder of the study. Feed consumption by females receiving 2500 ppm averaged 27 % less than that by the controls during most of the study.

The absolute and relative liver weights of males and females exposed to 2500 ppm of the test material were slightly but significantly greater than those of the controls. Males exposed to 500, 1000 or 2500 ppm and females exposed to 2500 ppm had significantly increased absolute and relative spleen weights. No clinical findings in mice were considered chemical related.

Haematocrit concentrations and erythrocyte counts of males receiving 1000 or 2500 ppm were significantly less than those of the controls; haemoglobin concentrations in males receiving 2500 ppm were significantly less and mean erythrocyte volume was significantly less in males receiving 2500 ppm. Females in the 1000 and 2500 ppm groups had significantly decreased haematocrit concentrations and erythrocyte counts; 2500 ppm females also had significantly decreased haemoglobin concentrations and mean erythrocyte volumes. Kupffer cell hypertrophy, bile duct hyperplasia, and an increase in size and number of macrophages in mesenteric lymph nodes were present in 2500 ppm male and female mice.

Under the conditions of this study, the NOAEL was 500 ppm (equivalent to 65 and 60 mg/kg bw/day in males and females, respectively) based on haematological effects at 1000 ppm and decreased body weight gain and effects on the liver at 2500 ppm.

 

2 Year Mouse Study: Groups of 80 male and 80 female mice were fed diets containing 0, 250, 500 or 1000 ppm of the test material for 104 weeks. Equivalent to ingested dose levels of 0, 30, 60 and 145 mg/kg bw/day in males and 0, 45, 110 and 225 mg/kg bw/day in females.

Two year survival rates were similar to those of the control and there were no clinical findings attributed to administration of the test material.

The final mean body weights of male and female mice exposed to 1000 ppm of the test material were 8 and 18 % lower than the controls, respectively. The final mean body weights of females exposed to 250 or 500 ppm were 8 to 9 % lower than that of the controls. Feed consumption by exposed males was similar to the controls.

A 15 month interim evaluation was conducted, at which point haematocrit level, haemoglobin concentration and erythrocyte count in 1000 ppm male mice were significantly lower than those in the controls. Serum alkaline phosphatase activities in 1000 ppm males were slightly but significantly greater than those in the controls at 3 and 9 months, as was the serum alkaline phosphatase activity in 1000 ppm females at 9 months. Serum levels of total bilirubin in all exposed groups of males were significantly greater than those in the controls at 9 and 15 months.

In the liver of male mice, negative trends in the incidences of fatty change, clear cell foci and adenoma or carcinoma combined occurred at the end of the study. There were no test material related increased incidences of neoplasms or non-neoplastic lesions.

Under the conditions of this study, the NOAEL was 500 ppm (equivalent to 60 mg/kg bw/day) based on decreased body weight gain and effects on the liver seen at 1000 ppm. There was no evidence of carcinogenic activity.

 

Based on the data provided, the structural analogue was shown to be more toxic that the registered substance in repeat dose studies; and would therefore represent the worst case scenario for the registered substance.

 

INHALATION TOXICITY

In accordance with point 8.6.1 and 8.6.2, column 1 and column 2 of Annexes VIII and IX respectively, of Regulation (EC) No. 1907/2006, testing for this endpoint should be performed using an appropriate route of exposure. A 28 day and 90 day oral study have been submitted to fulfil the subacute and subchronic toxicity of the registered substance. Exposure via the oral route is considered to be the most appropriate route of exposure as the physical chemical properties such as the physical state (liquid at room temperature and pressure) and vapour pressure (< 0.0015 Pa at 20 °C) indicate that exposure via inhalation is unlikely. Testing for this endpoint via the inhalation route is therefore omitted on this basis.

DERMAL TOXICITY

In accordance with point 8.6.1 and 8.6.2, column 1 and column 2 of Annexes VIII and IX respectively, of Regulation (EC) No. 1907/2006, testing for this endpoint should be performed using an appropriate route of exposure. Subacute and subchronic toxicity have already been sufficiently addressed in a 28 day and 90 day oral toxicity study submitted on the registered substance. Furthermore, findings from acute dermal toxicity testing indicate no grounds for concern. Repeated dose testing via the dermal route has therefore been omitted.


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
The repeat dose toxicity of the registered substance (2,2'-Methylenebis(6-nonyl-p-cresol)) was determined in a 90 day oral toxicity study conducted in accordance with generally accepted scientific in compliance with agreed protocols. The study was reported to a good standard with a sufficient level of detail to assess the quality of the submitted data. The study was assigned a reliability score of 1 in line with the principles for assessing data quality as defined by Klimisch (1997).

Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:
A data waiver has been submitted to address this endpoint.

Justification for selection of repeated dose toxicity inhalation - local effects endpoint:
A data waiver has been submitted to address this endpoint.

Justification for selection of repeated dose toxicity dermal - systemic effects endpoint:
A data waiver has been submitted to address this endpoint.

Justification for selection of repeated dose toxicity dermal - local effects endpoint:
A data waiver has been submitted to address this endpoint.

Justification for classification or non-classification

According to the criteria outlined in Regulation (EC) No. 1272/2008 and Directive 67/548/EEC, this substance does not meet the criteria for classification for repeated dose toxicity.