Registration Dossier

Administrative data

Description of key information

Repeated Dose Toxicity: Subchronic (90 days) study oral (gavage), rats (Sprague-Dawley) m/f (EPA OPP 82-1): NOAEL = 20 mg/kg bw/d (males), 40 mg/kg bw/d (females) (based on body weight and weight gain)

Repeated Dose Toxicity: Subacute (28 days) study oral (gavage), rats (Sprague-Dawley) m/f (equivalent to OECD 407): NOAEL = 50 mg/kg bw/d (based on haematology, clinical biochemistry, histopathology)

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

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)
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
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
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
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
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.
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
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
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:
kidney
liver
thyroid gland
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified

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.

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.

Endpoint:
short-term repeated dose 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)
Qualifier:
no guideline followed
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Version / remarks:
The study was conducted as a range-finding experiment to support subchronic oral testing in rats under US EPA FIFRA § 82-1.
Principles of method if other than guideline:
The study was conducted as a range-finding experiment to support subchronic oral testing in rats under US EPA FIFRA § 82-1.
GLP compliance:
yes
Remarks:
self-certified to US EPA regulations [40 CFR Part 160] and OECD regulations [C(81) 30 (Final) Annex 2]
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Remarks:
Sprague-Dawley Crl:CD®BR rats
Details on species / strain selection:
n/a
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
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
VEHICLE
- Amount of vehicle (if gavage): 5 ml/kg
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
28 days
Frequency of treatment:
daily
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
control
Dose / conc.:
50 mg/kg bw/day (nominal)
Dose / conc.:
250 mg/kg bw/day (nominal)
Dose / conc.:
500 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: purpose of the study: study was conducted as a range-finding experiment to support subchronic oral testing in rats
Positive control:
not required
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 prior to necropsy.

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: Not specified

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the time of necropsy

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the time of necropsy
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
Complete necropsies were performed on all rats that were found dead and at study termination. Selected organs were weighed at the scheduled necropsy.

HISTOPATHOLOGY: Yes
Selected tissues were examined microscopically from all animals.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
All top dose animals were found dead between study days 2 and 3. Ethoxyquin-related clinical signs in the 1,000 mg/kg bw/day group included salivation and yellow matting on various body surfaces. Test article-related gross lesions were observed in the gastrointestinal tract, kidneys, and lymph nodes. Histopathological lesions in the 1,000 mg/kg bw/day group were seen in the kidneys, liver, lungs, stomach, and lymph nodes.
All animals in the groups receiving 50, 250 and 500 mg/kg bw/day survived to the scheduled necropsy on study day 28. Test article-related clinical signs consisted of infrequent occurrences of salivation, yellow matting on various body surfaces, and/or brown urine in the 250 and 500 mg/kg bw/day. These signs were observed in a dose-and time-related manner at the time of dosing, about one hour later, and/or at the detailed physical examination prior to necropsy.
Mortality:
mortality observed, treatment-related
Description (incidence):
All top dose animals were found dead between study days 2 and 3. The cause of death for two of these animals was described histopathologically as mucosal necrosis and ulceration of the forestomach; while mortality in the remaining animals in this group may have been related to kidney and/or forestomach changes. All of these deaths were considered to be test article related.
All animals in the groups receiving 50, 250 and 500 mg/kg bw/day survived to the scheduled necropsy on study day 28.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Mean body weight gain in the 500 mg/kg bw/day group males was decreased during study week 0 to 1 and in the 500 mg/kg bw/day group females generally throughout the dosing period.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Mean food consumption in the 500 mg/kg bw/day group was slightly decreased during study week 0 to 1.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not specified
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Red blood cell, haemoglobin, and haematocrit means were decreased in the 250 mg/kg bw/day group females and the 500 mg/kg bw/day group males and females. Test article related changes in serum chemistry parameters were observed in the 250 and 500 mg/kg bw/day group animals and were more extensive in males than females.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Changes in the 500 mg/kg bw/day group consisted of increased albumin, globulin, total protein, bilirubin, urea nitrogen, gamma glutamyltransferase, cholesterol, calcium, phosphorus, and potassium means and decreased values for glucose and A/G ratio. The 250 mg/kg bw/day group had increased mean globulin, total protein, calcium, phosphorus, potassium, and cholesterol values and a decreased A/G ratio.
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 weights (absolute and relative to final body weight) in the 250 and 500 mg/kg bw/day group males and females were higher than the control group values at scheduled necropsy.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Test article-related microscopic lesions were observed in the kidneys, liver, lungs, stomach, and mediastinal lymph nodes of the 500 mg/kg bw/day group animals. Kidney lesions observed in the 500 mg/kg bw/day group included tubular dilatation in all males and four females, regeneration of the tubular epithelium in all males and four females, lymphocytic infiltration in four males and three females, and hyperplasia of the urothelium in a single female. The only liver lesion noted in the 500 mg/kg bw/day group animals was hepatocellular swelling in two females. Pulmonary lesions in the 500 mg/kg bw/day group animals included alveolar edema in a single male, and haemorrhage in two males and three females. Stomach lesions in the 500 mg/kg bw/day group were limited to a single female and included squamous cell hyperplasia, mononuclear leukocytic infiltration, and submucosal edema. Congestion of the mediastinal lymph node was observed in a single 500 mg/kg bw/day group female.
In the 250 mg/kg bw/day group, test article-related lesions were observed in the kidneys and included regeneration of the tubular epithelium in three males and renal tubular dilatation in two males. The low incidence of renal lymphocytic infiltration in the 50 and 250 mg/kg bw/day groups was not considered to be treatment-related.
No other ethoxyquin-related lesions were observed microscopically. Other histopathologic lesions were observed at a similar incidence in the control group (lymphocytic infiltration of the heart, nonsuppurative inflammation of the liver, etc.) or were seen at low incidence, typically in single animals (hydronephrosis, nonsuppurative inflammation of the lungs).
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Test article-related microscopic lesions were observed in the kidneys, liver, lungs, stomach, and mediastinal lymph nodes of the 500 mg/kg bw/day group animals. Kidney lesions observed in the 500 mg/kg bw/day group included tubular dilatation in all males and four females, regeneration of the tubular epithelium in all males and four females, lymphocytic infiltration in four males and three females, and hyperplasia of the urothelium in a single female. The only liver lesion noted in the 500 mg/kg bw/day group animals was hepatocellular swelling in two females. Pulmonary lesions in the 500 mg/kg bw/day group animals included alveolar edema in a single male, and haemorrhage in two males and three females. Stomach lesions in the 500 mg/kg bw/day group were limited to a single female and included squamous cell hyperplasia, mononuclear leukocytic infiltration, and submucosal edema. Congestion of the mediastinal lymph node was observed in a single 500 mg/kg bw/day group female.
In the 250 mg/kg bw/day group, test article-related lesions were observed in the kidneys and included regeneration of the tubular epithelium in three males and renal tubular dilatation in two males. The low incidence of renal lymphocytic infiltration in the 50 and 250 mg/kg bw/day groups was not considered to be treatment-related.
No other ethoxyquin-related lesions were observed microscopically. Other histopathologic lesions were observed at a similar incidence in the control group (lymphocytic infiltration of the heart, nonsuppurative inflammation of the liver, etc.) or were seen at low incidence, typically in single animals (hydronephrosis, nonsuppurative inflammation of the lungs).
Histopathological findings: neoplastic:
not specified
Other effects:
not specified
Key result
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: n.a.
Dose descriptor:
LOAEL
Effect level:
250 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
haematology
clinical biochemistry
histopathology: non-neoplastic
Dose descriptor:
LOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
mortality
Critical effects observed:
yes
Lowest effective dose / conc.:
500 mg/kg bw/day (nominal)
System:
other: no single system can be specified due to the variety of effects
Organ:
kidney
liver
lungs
lymph node
stomach
Treatment related:
yes
Dose response relationship:
not specified
Relevant for humans:
not specified

Table Clinical Chemistry-Selected Parameters(+SD)

Parameter

Sex

Dose (mg/kg bw/day)

0 (vehicle)

50

250

500

Albumin (g/dL)

M

4.4 (0.22)

4.7(0.24)

4.7 (0.17)

4.5 (0.37)

F

4.8 (0.19)

5.2 (0.69)

5.0 (0.25)

5.7* (0.12)

Protein (g/dL)

M

6.2 (0.25)

6.5 (0.36)

6.9* (0.25)

6.9* (0.43)

F

6.6 (0.24)

6.9 (0.59)

7.0 (0.29)

7.9* (0.26)

Globulin (g/dL)

M

1.8 (0.23)

1.8 (0.18)

2.1* (0.13)

2.4** (0.18)

F

1.8 (0.25)

1.7 (0.29)

2.0 (0.11)

2.2 (0.22)

A/G ratio

M

2.55 (0.463)

2.57 (0.254)

2.24 (0.129)

1.89** (0.197)

F

2.64 (0.495)

3.20 (0.979)

2.57 (0.186)

2.64 (0.271)

Total Bilirubin (mg/dL)

M

0.1 (0.04)

0.2 (0.04)

0.2* (0.04)

0.3** (0.05)

F

0.4 (0.29)

0.3 (0.09)

0.3 (0.00)

0.8 (0.50)

Urea nitrogen (mg/dL)

M

10.1 (0.53)

12.6 (1.35)

12.3 (1.97)

15.4** (2.20)

F

16.3 (3.96)

11.7 (0.91)

16.1 (1.08)

16.2 (4.77)

Alkaline phosphatase (IU/L)

M

179 (35.8)

188 (66.8)

158 (32.5)

149 (33.3)

F

139 (32.4)

102 (25.7)

69** (5.0)

77** (20.0)

Gamma glutamyl-transferase (IU/L)

M

0 (0.0)

0 (0.0)

1 (0.5)

8** (5.1)

F

0 (0.0)

0 (0.4)

0 (0.5)

11** (8.1)

Glucose (mg/dL)

M

214 (24.2)

163 (38.6)

189 (59.8)

134* (23.8)

F

145 (22.3)

165 (62.7)

137 (18.7)

144 (26.0)

Cholesterol (mg/dL)

M

66 (6.7)

66 (12.7)

96** (15.1)

116** (14.2)

F

68 (17.2)

88 (36.9)

106 (20.4)

177** (30.4)

Calcium (mg/dL)

M

11.3 (0.33)

11.3 (0.69)

12.2* (0.16)

12.4** (0.37)

F

11.8 (0.68)

12.2 (0.63)

12.3 (0.98)

12.3 (0.97)

Phosphorus (mg/dL)

M

10.8 (0.40)

10.5 (0.48)

10.9 (0.37)

11.6* (0.29)

F

11.4 (1.64)

10.5 (1.15)

10.8 (0.86)

10.6 (1.07)

Potassium (mEq/L)

M

7.89 (0.607)

8.03 (0.527)

7.77 (0.488)

9.66** (0.846)

F

8.67 (0.485)

8.19 (0.515)

8.31 (0.287)

8.44 (0.592)

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

All values are the means for five animals, with the exception of the 500 mg/kg bw/day females, with only four animals included. Top dose animals receiving 1000 mg/kg/day could not be analysed because of premature death.

Conclusions:
As information was provided via a peer-reviewed assessment report, it can be considered as sufficiently reliable to assess the subacute toxicity of ethoxyquin towards rats. Available data was gained within a range-finding study for a 90 d study.
As no treatment-related effects were observed at 50 mg/kg bw/day, the NOAEL was set as 50 mg/kg bw/day. In the next higher dose group, treatment-related effects were observed, leading to a LOAEL=250 mg/kg bw/day based on:
Red blood cell, haemoglobin, and haematocrit were decreased in the 250 mg/kg bw/day group females and the 500 mg/kg bw/day group males and females. Test article related changes in serum chemistry parameters were observed in the 250 and 500 mg/kg bw/day group animals and were more extensive in males than females. Test article-related histopathologic lesions were observed in the kidneys, liver, lungs, stomach, and lymph nodes of the 500 mg/kg bw/day group animals and in the kidneys of the 250 mg/kg bw/day group animals.
Effects noted at 1,000 mg/kg bw/day: death of all animals within 2-3 days.
According to Regulation 1272/2008, guidance values for classification as STOT RE Cat. 2 are 10 < LOAEL < 100 mg/kg for subchronic studies, hence, for a subacute one it would be 30 < LOAEL < 300 mg/kg. Despite the fact that effects were observed at 250 mg/kg bw, those are not distinct enough to indicate a specific organ toxicity, or to exclude an adaptive response, which may not necessarily be considered as adverse (regeneration of the tubular epithelium in three males and renal tubular dilatation in two males). Further, only the kidneys of male rats are affected, leading to the conclusion that this effect is not necessarily relevant for humans at all due to a very high species- and sex-specificity for renal toxicity.
Consequently, the results of this study do not trigger classification as STOT RE.
Executive summary:

The toxicity of ethoxyquin was evaluated in a 28-day oral range finding study in rats. The test article in the vehicle (corn oil) was administered orally by gavage to four groups of five male and five female 6 week old Sprague-Dawley Crl:CD®BR rats. Dosage levels were 0, 50, 250, 500 and 1,000 mg/kg bw/day.

The NOAEL was found to be 50 mg/kg bw/d.

The LOAEL=250 mg/kg bw/day was based on: Red blood cell, haemoglobin, and haematocrit were decreased in the 250 mg/kg bw/day group females and the 500 mg/kg bw/day group males and females. Test article-related changes in serum chemistry parameters were observed in the 250 and 500 mg/kg bw/day group animals and were more extensive in males than females. Test article-related histopathologic lesions were observed in the kidneys, liver, lungs, stomach, and lymph nodes of the 500 mg/kg bw/day group animals and in the kidneys of the 250 mg/kg bw/day group animals.

Effects noted at 1,000 mg/kg bw/day: death of all animals within 2-3 days.

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

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
20 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
There are two repeated dose toxicity studies on ethoxyquin available, ranging from subacute (28 days) to subchronic (90 days) in rats. All studies were properly conducted equivalent to the respective guidelines, and are sufficiently documented. According to REACH Annex IX, a subchronic study is required to cover this endpoint, which is available. Hence, the data requirements under REACH are fully met, no further testing is required. There is no indication given that the data is not relevant for humans. So, the data base is of a very good quality.

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

The available study results do not allow a Mode of Action Analysis. The 90 day study in rats identified adverse effects on haematology and clinical chemistry parameters, thyroid glands, and abnormal urinary bladder contents (400 mg/kgbw/day group only), increased liver and kidney weight means (absolute and relative), and microscopic kidney lesions. In the 28 day study in rats, red blood cell count, haemoglobin, and haematocrit were decreased in the 250 mg/kg bw/day group females and the 500 mg/kg bw/day group males and females. Test article-related changes in serum chemistry parameters were observed in the 250 and 500 mg/kg bw/day group animals and were more extensive in males than females. Test article-related histopathologic lesions were observed in the kidneys, liver, lungs, stomach, and lymph nodes of the 500 mg/kg bw/day group animals and in the kidneys of the 250 mg/kg bw/day group animals.

In conclusion, there was not a consistent specific effect observed in all studies or species. So there is also no indication given that the data is not relevant for humans and every effect may also not be observed in humans.

Additional information

Justification for classification or non-classification

In the 28 day study in rats, red blood cell count, haemoglobin, and haematocrit were decreased in the 250 mg/kg bw/day group females and the 500 mg/kg bw/day group males and females. Test article related changes in serum chemistry parameters were observed in the 250 and 500 mg/kg bw/day group animals and were more extensive in males than females. Test article-related histopathologic lesions were observed in the kidneys, liver, lungs, stomach, and lymph nodes of the 500 mg/kg bw/day group animals and in the kidneys of the 250 mg/kg bw/day group animals.

Effects noted at 1,000 mg/kg bw/day: death of all animals within 2-3 days.

According to Regulation 1272/2008, guidance values for classification as STOT RE Cat. 2 are 10 < LOAEL < 100 mg/kg for subchronic studies, hence, for a subacute one it would be 30 < LOAEL < 300 mg/kg. Despite the fact that effects were observed at 250 mg/kg bw, those are not distinct enough to indicate a specific organ toxicity, or to exclude an adaptive response, which may not necessarily be considered as adverse (regeneration of the tubular epithelium in three males and renal tubular dilatation in two males). Further, only the kidneys of male rats are affected, leading to the conclusion that this effect is not necessarily relevant for humans at all due to a very high species- and sex-specificity for renal toxicity.

Consequently, the results of this study do not trigger classification as STOT RE.

 

In the 90 day study in 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.

 

So in summary, there is not trigger given for the classification of ethoxyquin as STOT RE according to Regulation 1272/2008.