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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
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Remarks:
Peer-reviewed assessment report (attached in section 13)

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
2007
Report Date:
2007

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
EPA OPP 82-1 (90-Day Oral Toxicity)
Version / remarks:
US EPA FIFRA Guideline § 82-1
Deviations:
yes
Remarks:
Test material purity and stability were not determined and were the responsibility of the sponsor.
Qualifier:
equivalent or similar to
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
Directive 88/302/EEC (OJ No L133/8 of 30 May 1988)
Deviations:
not applicable
GLP compliance:
yes
Remarks:
self-certified to US EPA regulations [40 CFR Part 160]; OECD regulations [C(81) 30 (Final) Annex 2]; and MAFF regulations [59 NohSan No. 3850]
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Chemical name
IUPAC: 6-ethoxy-2,2,4-trimethyl-1,2-dihydroquinoline
CAS: 6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline

Test animals

Species:
rat
Strain:
Sprague-Dawley
Remarks:
Sprague-Dawley Crl:CD®BR rats
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories Inc., Portage Ml USA
- Age at study initiation: 6 weeks

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
VEHICLE
- Amount of vehicle (if gavage): 5 ml
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
90 days
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
negative control, vehicle (Mazola® corn oil)
Dose / conc.:
20 mg/kg bw/day (nominal)
Remarks:
determined based on a 28-day oral range finding study in rats
Dose / conc.:
40 mg/kg bw/day (nominal)
Remarks:
determined based on a 28-day oral range finding study in rats
Dose / conc.:
200 mg/kg bw/day (nominal)
Remarks:
determined based on a 28-day oral range finding study in rats
Dose / conc.:
400 mg/kg bw/day (nominal)
Remarks:
determined based on a 28-day oral range finding study in rats
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dosage levels were determined based on a 28-day oral range finding study in rats
Positive control:
not required

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: The animals were observed twice daily for signs of mortality and moribundity.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Clinical examinations were performed daily and detailed physical examinations were performed weekly.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were recorded weekly.

FOOD CONSUMPTION AND COMPOUND INTAKE:
Food consumption was recorded weekly.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Ophthalmological examinations were conducted on all animals prior to the initiation of dosing and during study week 12.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: performed at necropsy.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: performed at necropsy.

URINALYSIS: Yes
- Time schedule for collection of urine: performed at necropsy.

NEUROBEHAVIOURAL EXAMINATION: Not specified

IMMUNOLOGY: Not specified
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
Complete necropsies were performed on all rats at study termination and selected organs were weighed.

HISTOPATHOLOGY: Yes
Selected tissues were examined microscopically from all animals.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Test article-related clinical signs were observed in the 200 and 400 mg/kg bw/day groups and consisted of yellow material on various body surfaces, salivation, red material around the mouth, and/or brown material on the urogenital area. These findings were typically more prevalent in females.
Mortality:
no mortality observed
Description (incidence):
All animals survived to the scheduled necropsy.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Mean body weight gains were reduced in the 40, 200 and 400 mg/kg bw/day group males essentially throughout the study and occasionally in the 200 and 400 mg/kg bw/day females from study weeks 9 through 13.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
The 200 and 400 mg/kg bw/day group males experienced a transient reduction in food consumption during the first week of dosing (study week 0 to 1).
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
Ophthalmology did not reveal remarkable findings that could be attributed to treatment.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Test article-related effects on haematology parameters at study termination consisted of decreased red blood cell count, haemoglobin, and haematocrit means in the 200 and 400 mg/kg bw/day group males and females, and increased mean reticulocyte counts in the 200 mg/kg bw/day group females and the 400 mg/kg bw/day group males and females.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Test article-related changes in serum clinical chemistry parameters were observed in the 200 and 400 mg/kg bw/day group animals and consisted of increased serum protein levels and calcium means in the females, and increased bilirubin, gamma glutamyltransferase, cholesterol and TSH (males only in the 200 mg/kg bw/day group) means. Mean T4 was decreased in the 400 mg/kg bw/day group males. In addition, mean urea nitrogen was increased and mean glucose was decreased in the 400 mg/kg bw/day group males and females. These changes, while slight, were considered to be potentially related to treatment. Discoloured urine was noted in the 200 and 400 mg/kg bw/day groups.
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Mean liver and kidney weights (absolute and relative to final body weight) were increased in the 200 and 400 mg/kg bw/day group males and females.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
At the macroscopic evaluation, reddened and/or enlarged thyroid glands were observed in the 200 and 400 mg/kg bw/day group males and females, and abnormal contents of the urinary bladder were noted in the 400 mg/kg bw/day group.
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
At the microscopic examination, ethoxyquin-related lesions were observed in the kidneys of the 200 and 400 mg/kg bw/day group animals. These findings comprised nephropathy in females, and papillary necrosis as well as hyaline droplets in both sexes.
Histopathological findings: neoplastic:
not specified

Effect levels

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
20 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
body weight and weight gain
Key result
Dose descriptor:
NOAEL
Effect level:
40 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
body weight and weight gain
Dose descriptor:
LOAEL
Effect level:
40 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
body weight and weight gain
Dose descriptor:
LOAEL
Effect level:
200 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
haematology
clinical biochemistry
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic

Target system / organ toxicity

Critical effects observed:
yes
Lowest effective dose / conc.:
200 mg/kg bw/day (nominal)
System:
other: no single system can be specified due to the variety of effects
Organ:
liver
kidney
thyroid gland
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified

Any other information on results incl. tables

Table Cumulative body weight gain (g) in rats as compared to pre-treatment (Week 0) values; absolute and (in brackets) relative (%) gain

Dose

Sex

Week 1

Week 3

Week 5

Week

Week 9

Week 11

Week 13

0

M

50 (6.5)

137 (21.4)

208 (35.5)

257 (46.0)

295 (50.0)

323 (53.1)

353 (57.8)

F

18 (4.5)

53 (6.7)

79 (6.4)

100 (13.2)

109 (12.3)

125 (13.7)

132 (17.7)

20

M

53 (5.7)

138 (22.0)

210 (36.7)

256 (46.8)

293 (53.9)

324 (61.1)

35.1 (65.3)

F

21 (4.0)

57 (7.6)

85 (11.4)

102 (14.3)

116 (17.0)

138 (17.9)

142 (14.8)

40

M

43* (4.1)

111* (15.5)

168* (22.7)

210* (28.3)

240* (31.0)

272 (36.1)

295* (40.8)

F

25* (8.1)

63 (16.7)

88 (21.9)

106 (26.5)

124 (26.4)

138 (30.4)

148 (31.5)

200

M

37** (6.6)

107** (20.6)

161** (31.5)

200** (34.7)

227** (38.3)

250** (40.0)

274** (42.2)

F

19 (4.3)

52 (11.2)

75 (11.9)

91 (15.9)

101 (15.9)

108 (19.0)

118 (21.3)

400

M

38** (6.0)

117 (15.6)

178 (30.4)

221 (31.5)

245* (34.2)

267* (35.1)

293* (38.5)

F

22 (5.6)

50 (10.5)

77 (13.8)

91 (16.1)

103 (15.4)

110 (17.6)

119 (20.1)

Notes: All dosages in mg/kg bw/day. Values represent the mean of 10 animals per sex.

Statistically significant from control group at (*) 0.05 or (**) 0.01 level using Dunnett's test.

 

Table Haematology-Selected Parameters(+SD)

Parameter

Sex

Dose (mg/kg bw/day)

0

20

40

200

400

WBC (103/µL)

M

14.8 (2.93)

14.0 (2.07)

13.1 (2.57)

12.3 (2.47)

11.7* (2.64)

F

10.6 (2.26)

10.3 (2.65)

10.8 (1.68)

10.3 (2.30)

7.4** (1.22)

RBC

(106/µL)

M

9.17 (0.502)

8.72 (0.245)

9.03 (0.504)

8.43** (0.475)

7.81** (0.362)

F

8.19 (0.516)

8.21 (0.352)

8.25 (0.838)

7.54 (0.588)

6.91** (0.522)

Haemoglobin

(g/dL)

M

16.0 (0.85)

15.1* (0.41)

15.4 (0.58)

14.6** (0.79)

13.1** (0.47)

F

15.4 (0.83)

15.3 (0.70)

15.3 (1.32)

14.0** (0.53)

12.8** (0.59)

Haematocrit

(%)

M

47.4 (2.36)

44.8* (1.07)

45.9 (2.05)

43.7** (2.44)

39.5** (1.33)

F

45.4 (2.57)

45.1 (1.79)

45.0 (3.89)

41.8* (2.05)

38.4** (1.90)

Prothrombin time (sec)

M

16.5 (0.53)

16.6 (0.86)

16.3 (0.38)

16.1 (0.47)

15.6** (0.66)

F

16.0 (0.61)

15.8 (0.73)

16.0 (0.62)

15.7 (0.71)

15.2* (0.31)

Reticulocyte

(%)

M

0.6 (0.46)

NA

NA

0.8 (0.37)

1.6** (0.80)

F

0.6 (0.33)

NA

NA

1.3* (0.58)

1.8** (0.66)

Notes: Statistically significant from control group at (*) 0.05 or (**) 0.01 level using Dunnett's test. All values are the means for ten animals. NA = Not applicable.

 

Table Clinical chemistry-Selected Parameters(+SD)

Parameter

Se

x

Dose (mg/kg bw/day)

0 (vehicle)

20

40

200

400

Albumin (g/dL)

M

4.6 (0.35)

4.6 (0.24)

4.7 (0.28)

4.8 (0.32)

4.5 (0.22)

F

5.1 (0.54)

5.1 (0.41)

5.2 (0.28)

5.6 (0.53)

5.7* (0.45)

Protein (g/dL)

M

7.0 (0.43)

6.8 (0.34)

7.0 (0.25)

7.3 (0.42)

6.9 (0.43)

F

7.1 (0.53)

7.2 (0.37)

7.2 (0.22)

7.9**(0.46)

7 9** (0.70)

Globulin (g/dL)

M

2.4 (0.14)

2.2 (0.14)

2.3 (0.20)

2.5 (0.18)

2.4 (0.25)

F

2.0 (0.13)

2.1 (0.14)

2.0 (0.26)

2.3* (0.31)

2.3* (0.27)

A/G ratio

M

1.94 (0.147)

2.06 (0.109)

2.02 (0.262)

1.93 (0.150)

1.91 (0.160)

F

2.61 (0.342)

2.46 (0.309)

2.62 (0.461)

2.50 (0.470)

2.52 (0.207)

Total bilirubin (mg/dL)

M

0.2 (0.05)

0.2 (0.05)

0.2 (0.05)

0.3* (0.08)

0.4** (0.05)

F

0.2 (0.05)

0.2 (0.05)

0.2 (0.06)

0.3** (0.05)

0.5** (0.08)

Urea nitrogen (mg/dL)

M

11.7 (2.29)

12.0 (1.60)

13.1 (3.81)

13.0 (2.20)

17.7** (3.87)

F

12.5 (2.17)

14.1 (3.82)

11.9 (1.90)

14.3 (3.46)

15.8 (3.29)

Gamma glutamyl-transferase (IU/L)

M

0 (0.4)

0 (0.4)

0 (0.3)

2 (0.8)

14** (5.5)

F

0 (0.5)

1 (0.9)

1 (0.7)

2 (1.3)

9** (4.7)

Glucose (mg/dL)

M

221 (46.3)

207 (19.1)

227 (37.8)

206 (45.6)

155** (38.1)

F

193 (27.9)

206 (27.0)

185 (35.0)

167 (34.5)

149** (15.8)

Cholesterol (mg/dL)

M

67 (15.7)

63 (20.4)

65 (17.7)

96* (23.7)

134** (29.2)

F

87 (14.4)

87 (18.2)

91 (19.5)

126** (26.2)

185** (37.3)

Calcium (mg/dL)

M

11.4 (0.41)

11.1 (0.29)

11.4 (0.39)

11.7 (0.48)

11.6 (0.25)

F

11.2 (0.60)

11.4 (0.41)

11.4 (0.36)

12.0** (0.31)

12.1** (0.35)

Notes: Statistically significant from control group at (*) 0.05 or (**) 0.01 level using Dunnett's test. All values are the means for ten animals.

Applicant's summary and conclusion

Conclusions:
As information was provided via a peer-reviewed assessment report, it can be considered as sufficiently reliable to assess the subchronic toxicity of ethoxyquin towards rats.
No signs of toxicity were noted in the 20 mg/kg bw/day group, so the NOAEL was set as 20 mg/kg bw/day. At a dose level of 40 mg/kg bw/day, the only test article-related effect was slightly decreased mean body weight gain in males.
In the 200 and 400 mg/kg bw/day groups, signs of toxicity consisted of changes in the clinical conditions of the animals, decreased mean body weight gains, a transient reduction in food consumption (males only), changes in haematology and clinical chemistry parameters, reddened and/or enlarged thyroid glands, abnormal urinary bladder contents (400 mg/kg bw/day group only), increased liver and kidney weight means (absolute and relative), and microscopic kidney lesions.
Based on these findings, the no observable adverse effect level (NOAEL) for system toxicity of ethoxyquin administered orally (by gavage) to rats for 90 days was 20 mg/kg bw/day for males and 40 mg/kg bw/day for females event though the only finding at 40 mg/kg bw/day in males was a slightly decreased mean body weight gain. This was done out of precautionary reasons.
According to Regulation 1272/2008, guidance values for classification as STOT RE Cat. 2 are 10 < LOAEL < 100 mg/kg for subchronic studies. As no organ-related adverse effects were noted at 40 mg/kg, and the next dosage level was way above the guidance values, the results of this study do not trigger classification as STOT RE.
Executive summary:

The toxicity of ethoxyquin was evaluated in a 90-day oral gavage study in rats. The test article in the vehicle (corn oil) was administered orally by gavage to four groups of ten male and ten female 6 week old Sprague-Dawley Crl:CD®BR rats. Dosage levels were 0, 20, 40, 200, and 400 mg/kg bw/day.

No signs of toxicity were noted in the 20 mg/kg bw/day group, sothe NOAEL was set as 20 mg/kg bw/day. At a dose level of 40 mg/kg bw/day, the only test article-related effect was slightly decreased mean body weight gain in males.

In the 200 and 400 mg/kg bw/day groups, signs of toxicity consisted of changes in the clinical conditions of the animals, decreased mean body weight gains, a transient reduction in food consumption (males only), changes in haematology and clinical chemistry parameters, reddened and/or enlarged thyroid glands, abnormal urinary bladder contents (400 mg/kgbw/day group only), increased liver and kidney weight means (absolute and relative), and microscopic kidney lesions.

Based on these findings, the no observable adverse effect level (NOAEL) for system toxicity of ethoxyquin administered orally (by gavage) to rats for 90 days was 20 mg/kg bw/day for males and 40 mg/kg bw/day for females event though the only finding at 40 mg/kg bw/day in males was a slightly decreased mean body weight gain.

The study was classified as acceptable. No necessity for classification according to Regulation 1272/2008 was identified.