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Toxicity to reproduction

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Administrative data

Endpoint:
one-generation reproductive toxicity
Remarks:
A Combined /Chronic Toxicity study/Carcinogenicity /study and one generation study, in which the animals were mated to produce an F1 litter to assess the reproductive/developmental toxicity. Less animals were used compared to the current OECD Guidelines
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1968
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
Not all the raw study results have been published in the publication, limited information available on methods, 1st generation included, but less animals were used.,
Cross-reference
Reason / purpose for cross-reference:
reference to same study
Reference
Endpoint:
chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1968
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
Not all the raw study results have been published in the publication, limited information available on methods (no guideline followed, no info regarding dosing preparation).
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)
Version / remarks:
1981, EFSA assignment
GLP compliance:
no
Remarks:
pre-GLP
Limit test:
no
Species:
rat
Strain:
other: Charles River weanling albino
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River
- Age at study initiation: weanling
- Housing: Individual
- Diet: Ground rat food mixed with the test substance
- Water (e.g. ad libitum): ad libitum

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Mixing appropriate amounts with (Type of food): Rockland Ground Rat Food
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
None
Duration of treatment / exposure:
2 years
Frequency of treatment:
Daily
Dose / conc.:
200 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
50 mg/kg bw/day (nominal)
No. of animals per sex per dose:
25
Control animals:
yes, concurrent no treatment
Details on study design:
10 males and 10 females were mated to produce two separate litters, in order to test reproductive toxicity (described in the endpoint on reproductive toxicity)
Positive control:
not applicable
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: 0, 3, 6, 9, 12, 18 and 24 months
- Anaesthetic used for blood collection: Not specified
- Animals fasted: No
- How many animals: 5 males and 5 females per group
- Parameters checked: Hemoglobin, hematocrit, red blood cells, white blood cells, differential count

URINALYSIS: Yes
- Time schedule for collection of urine: 0, 3, 6, 9, 12, 18 and 24 months
- Metabolism cages used for collection of urine: Yes
- Animals fasted: overnight water deprivation, not fasted.
- Parameters checked: color, volume, specific gravity, pH, blood, albumin, glucose, and microscopic examination of the sediment after centrifugation

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

Sacrifice and pathology:
GROSS PATHOLOGY: Yes
The following organs were evaluated: heart, lung, liver, kidney, pancreas, spleen, thymus, mesenteric lymph node, adrenals, thyroid, brain, hypophysis, uterus and ovary;

HISTOPATHOLOGY: Yes
The following tissues were evaluated: brain (sectioned at the optic chiasm, mammillary body, cerebellum, pons, and medulla oblongata), cervical spinal cord, hypophysis, eye, parotid gland, thyroid and parathyroid, thymus, heart, lung, sternum, rib, aorta, liver, spleen, pancreas, kidneys, adrenals, stomach (fore and hind), small and large intestine (four levels), mesenteric lymph node, reproductive tract (all levels), urinary bladder, skeletal muscle, femoral nerve, femoral bone marrow, skin and mammary gland
Statistics:
No data
Clinical signs:
no effects observed
Description (incidence and severity):
All parameters were considered normal.
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
The body weight of test and control animals evolved in a comparable manner over the 2 year exposure period
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
All parameters were considered normal.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
One control rat developed anemia (RBC, 1.8 x 10^3 /mm3; hemoglobin, 8.0 % and hematocrit,18.5 %) of unknown etiology.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
All parameters were considered normal.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
All rats showed a tendency toward albuminuria; All other parameters were considered normal.
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
All parameters were considered normal.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Pathologic changes consistent with aged rats were observed primarily in the pulmonary, endocrine, urinary, and cardiovascular systems.
Neuropathological findings:
not examined
Description (incidence and severity):
No specific neurotoxicity examination was performed.
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Pathologic changes consistent with aged rats were observed primarily in the pulmonary, endocrine, urinary, and cardiovascular systems.
Histopathological findings: neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Neoplasia occurred in a random manner with no apparent relationship between number, location, or type of tumors and treatment. This was due presumably to the advanced age of the animals.
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Ethyl maltol had no effect on fertility, gestation, parturition, lactation, or fetal development. A fall in fertility was noted in test and control groups at the second mating. This was due presumably to the advanced age of the animals.
Key result
Dose descriptor:
NOAEL
Effect level:
>= 200 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: highest dose tested
Key result
Critical effects observed:
no
Conclusions:
This chronic oral toxicity study (feeding) in rats (similar to OECDTG 453) did not report any treatment-related effects. Based on these findings, a NOAEL of >=200 mg/kg bw/day was established.
Executive summary:

A chronic (2 year) repeated-dose toxicity study was performed which included the examination of carcinogenicity evaluation was performed similar to OECD TG 453. The study was rated Klimisch 2, as the study results were sufficiently described, but did not contain detailed descriptions of the individual animal data. Rats were given the test substance mixed into the diet at dose levels of 50, 100 or 200 mg/kg bw/day. A control group was included that received normal feed. In each dose group 25 male and 25 female animals were included. Observations regarding body weight, and food consumption were made weekly. Haematological and urinary parameters were measured after 0, 3, 6, 9, 12, 18 and 24 months. Blood glucose evaluations were omitted. Between 15 and 21 and 30 and 36 weeks, 10 males and 10 females per level were mated to produce two separate litters. The resulting offspring were counted, weighed, and examined for abnormal development at birth and during lactation. At weaning they were sacrificed and examined for internal malformations. In the parent groups, 5 of each sex at each level were autopsied after 1 year on test, the remainder after 2 years. At termination further examinations were made for gross necropsy, organ weights and tissue histology.

Exposure to Ethyl Maltol did not affect the life-span of the animals. No differences in body weight development were observed between control and treated groups. One control rat developed anemia after 2 years, but this was not considered treatment related. Hematology results for all other animals were normal. Urinalysis was normal except for the tendency for non-treatment related albuminuria in both control and treated rats. The substance had no effect on fertility, gestation, parturition, lactation, or fetal development, although a fall in fertility was noted in test and control groups at the second mating. This was due presumably to the advanced age of the animals. The observed pathological effects and neoplasia in the animals were also consistent with aged rats, and occurred in a random non treatment related manner. Based on these findings, a NOAEL of >=200 mg/kg bw/day was established.

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
1968

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]
Deviations:
yes
Remarks:
This is a combined one generation / chronic study and less animals than currently requested are used
GLP compliance:
no
Remarks:
pre-GLP
Limit test:
no

Test material

1
Chemical structure
Reference substance name:
2-ethyl-3-hydroxy-4-pyrone
EC Number:
225-582-5
EC Name:
2-ethyl-3-hydroxy-4-pyrone
Cas Number:
4940-11-8
Molecular formula:
C7H8O3
IUPAC Name:
2-ethyl-3-hydroxy-4H-pyran-4-one
Test material form:
solid

Test animals

Species:
rat
Strain:
other: Charles River Weanling Albino
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River
- Age at study initiation: weanling
- Housing: Individual
- Diet: Ground rat food mixed with the test substance
- Water (e.g. ad libitum): ad libitum

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
DIET PREPARATION
- Mixing appropriate amounts with (Type of food): Rockland Ground Rat Food
Details on mating procedure:
- Any other deviations from standard protocol:
Between 15 and 21 and 30 and 36 weeks, 10 males and 10 females per level were mated to pro-duce two separate litters.
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
No
Duration of treatment / exposure:
2 years
Frequency of treatment:
daily
Details on study schedule:
- Age at mating of the mated animals in the study: 15-21 and 30-36 weeks
Doses / concentrationsopen allclose all
Dose / conc.:
200 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
50 mg/kg bw/day (nominal)
No. of animals per sex per dose:
25
Control animals:
yes, concurrent no treatment

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

FOOD EFFICIENCY: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: 3, 6, 9, 12, 18 and 24 months
- Anaesthetic used for blood collection: Not specified
- Animals fasted: No
- How many animals: 5 males and 5 females per group
- Parameters checked: Hemoglobin, hematocrit, red blood cells, white blood cells, differential count

URINALYSIS: Yes
- Time schedule for collection of urine: 3, 6, 9, 12, 18 and 24 months
- Metabolism cages used for collection of urine: Yes
- Animals fasted: overnight water deprivation, not fasted.
- Parameters checked: color, volume, specific gravity, pH, blood, albumin, glucose, and microscopic examination of the sediment after centrifugation

Oestrous cyclicity (parental animals):
Not examined
Sperm parameters (parental animals):
Not examined
Litter observations:
STANDARDISATION OF LITTERS: No

PARAMETERS EXAMINED
The following parameters were examined in [F1] offspring:
number and sex of pups, presence of gross anomalies, weight gain. At weaning they were sacrificed and examined for internal malformations.

GROSS EXAMINATION OF DEAD PUPS:
not specified

Postmortem examinations (parental animals):
GROSS PATHOLOGY: Yes
The following organs were evaluated: heart, lung, liver, kidney, pancreas, spleen, thymus, mesenteric lymph node, adrenals, thyroid, brain, hypophysis, uterus and ovary;

HISTOPATHOLOGY: Yes
The following tissues were evaluated: brain (sectioned at the optic chiasm, mammillary body, cerebellum, pons, and medulla oblongata), cervical spinal cord, hypophysis, eye, parotid gland, thyroid and parathyroid, thymus, heart, lung, sternum, rib, aorta, liver, spleen, pancreas, kidneys, adrenals, stomach (fore and hind), small and large intestine (four levels), mesenteric lymph node, reproductive tract (all levels), urinary bladder, skeletal muscle, femoral nerve, femoral bone marrow, skin and mammary gland
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring was sacrificed at weaning
- These animals were subjected to postmortem examinations

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations

Statistics:
Not specified
Reproductive indices:
Calculation method not specified
Offspring viability indices:
Calculation method not specified

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Description (incidence and severity):
All parameters were considered normal.
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
The body weight of test and control animals evolved in a comparable manner over the 2 year exposure period
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
All parameters were considered normal.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
One control rat developed anemia (RBC, 1.8 x 103 /mm3; hemoglobin, 8.0g % and hematocrit,18.5 %) of unknown etiology.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
All parameters were considered normal.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
All rats showed a tendency toward albuminuria; All other parameters were considered normal.
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Pathologic changes consistent with aged rats were observed primarily in the pulmonary, endocrine, urinary, and cardiovascular systems.
Histopathological findings: neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Neoplasia occurred in a random manner with no apparent relationship between number, location, or type of tumors and treatment. This was due presumably to the advanced age of the animals.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, non-treatment-related
Description (incidence and severity):
A fall in fertility was noted in test and control groups at the second mating. This was due presumably to the advanced age of the animals.

1st mating P0
0 mg/kg bw/day 10% conception
50 mg/kg bw/day 90% conception
100 mg/kg bw/day 80% conception
200 mg/kg bw/day 80% conception

2nd mating P0
0 mg/kg bw/day 70% conception
50 mg/kg bw/day 60% conception
100 mg/kg bw/day 60% conception
200 mg/kg bw/day 50% conception

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Effect level:
>= 200 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: highest dose tested

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
1st F1 litter
0 mg/kg bw/day 93% survival
50 mg/kg bw/day 89% survival
100 mg/kg bw/day 91% survival
200 mg/kg bw/day 92% survival

2nd F1 litter
0 mg/kg bw/day 49% survival
50 mg/kg bw/day 58% survival
100 mg/kg bw/day 39% survival
200 mg/kg bw/day 66% survival
Body weight and weight changes:
no effects observed
Description (incidence and severity):
All parameters were considered normal.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
no effects observed
Description (incidence and severity):
All parameters were considered normal.
Histopathological findings:
not examined
Other effects:
not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined
Description (incidence and severity):
Not specifically examined, however also no abnormalities were reported

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
>= 200 mg/kg bw/day
Based on:
test mat.
Remarks:
not directly exposed (parental exposure only)
Sex:
male/female
Basis for effect level:
other: highest dose tested

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Applicant's summary and conclusion

Conclusions:
This combined one-generation/chronic oral toxicity study (feeding) in rat (similar to OECDTG 415) did not report any treatment-related reproductive effects. Based on these findings, a NOAEL of >=200 mg/kg bw/day was established.
Executive summary:

A combined one-generation and chronic (2 year) repeated dose toxicity study was performed similar to OECD TG 415/453 but with less animals. The study was rated Klimisch 2, as the study results were sufficiently described, but did not contain detailed descriptions of the individual animal data. Rats were given the test substance mixed into the diet at dose levels of 50, 100 or 200 mg/kg bw/day. A control group was included that received normal feed. In each dose group 25 male and 25 female animals were included. Observations regarding body weight, and food consumption were made weekly. Haematological and urinary parameters were measured after 3, 6, 9, 12, 18 and 24 months. Blood glucose evaluations were omitted. Between 15 and 21 and 30 and 36 weeks, 10 males and 10 females per level were mated to produce two separate litters. The resulting offspring were counted, weighed, and examined for abnormal development at birth and during lactation. At weaning they were sacrificed and examined for internal malformations. In the parent groups, 5 of each sex at each level were autopsied after 1 year on test, the remainder after 2 years. At termination further examinations were made for gross necropsy, organ weights and tissue histology.

Exposure to ethyl maltol did not affect the life-span of the animals. No differences in body weight development were observed between control and treated groups. One control rat developed anemia after 2 years, but this was not considered treatment releated. Hematology results for all other animals were normal. Urinalysis was normal except for the tendency for non-treatment related albuminuria in both control and treated rats.

Fertility and develomental toxicity: Ethyl maltol had no effect on fertility, gestation, parturition, lactation, or fetal development, although a fall in fertility was noted in test and control groups at the second mating. This was due presumably to the advanced age of the animals. The observed pathological effects and neoplasia in the animals were also consistent with aged rats, and occurred in a random non treatment related manner. Based on the results of this study, a reproductive NOAEL is set at >=200 mg/kg bw/day, since no adverse effects observed at highest dose tested.