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Toxicity to reproduction

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Endpoint:
toxicity to reproduction
Remarks:
other: chronic two-year study
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1957
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: No GLP (study of 1957), short description of results, few organs examined, unclear number of animals examined in histopathology, only one dose tested in females, purity not specified.
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:
1957
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: No GLP (study of 1957), short description of results, few organs examined, unclear number of animals examined, only one dose tested in females, purity not specified.
Principles of method if other than guideline:
Rats were fed either the basal laboratory diet, or the basal diet to which adipic acid was added. Body weights, food consumption, and general appearance were recorded weekly throughout the experimental period. Whenever possible, gross autopsy was performed on those animals that died during the course of the experiment. After two years, surviving rat were weighed, killed, and examined grossly. The brain, thyroid, lung, heart, liver, spleen, kidneys and adrenals, stomach of approximately half of each group of males were weighed. The kidneys, spleen, liver and heart of each female were weighed. Microscopic examination of thyroid, lung, heart, liver, spleen, kidneys, adrenals, stomach, pancreas, bone marrow, large and small intestine and testis or ovaries and uterus on a representative number of animals was performed.
GLP compliance:
no
Species:
rat
Strain:
other: Carworth Farm strain
Sex:
male/female
Details on test animals or test system and environmental conditions:
weight at study initiation: 50-60 g
housing individually in cages with wire mesh floors
free access to food and water
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency):
- Mixing appropriate amounts with (Type of food): basal diet
Duration of treatment / exposure:
2 years
Frequency of treatment:
diet ad libitum
Dose / conc.:
0.1 other: % in diet (approx. 75 mg/kg bw/day)
Remarks:
Dose / concentration for males
Dose / conc.:
1 other: % in diet (approx. 750 mg/kg bw/day)
Remarks:
Dose /concentration for males/females
Dose / conc.:
3 other: % in diet (appros. 2250 mg/kg bw/day)
Remarks:
Dose / concentration for males
Dose / conc.:
5 other: % in diet (approx. 3750 mg/kg bw/day)
Remarks:
Dose / concentration for males
No. of animals per sex per dose:
started with 20 males per treatment groups and in the control group
started with 19 females in the treatment group and 10 females in the control group
Control animals:
yes, plain diet
Positive control:
not applicable
Observations and examinations performed and frequency:
Body weights, food consumption and general appearance were recorded weekly throughout the experimental period.
Sacrifice and pathology:
After 2 years, surviving rats were weighed, sacrificed, and examined grossly. The brain, thyroid, lung, heart, liver, spleen, kidneys, adrenals, stomach and testes of the animals were weighed. Microscopic examination of thyroid, lung, heart, liver, spleen, kidneys, adrenals, stomach, pancreas, bone marrow, large and small intestine, uterus, ovaries and testes on a representative number of animals (no further information) was performed.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
The following signs were observed among all male groups, including the controls, especially during the final six months: wheezing, blood-tinged crust about the noses and eyes, and body sores. These findings were not significantly different among the groups, although a lower incidence of signs indicative of respiratory infection and body sores occurred in the 5% adipic acid group. 
Description (incidence):
The per cent survival was not negatively affected by treatment.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Growth of males treated with 0.1 and 1% and females treated with 1% test item in food was comparable to that of the respective controls. During the rapid growth period of the 2-year feeding studies, weight gains for the male rats receiving 3 or 5% adipic acid was significantly less (up to minus > 30%) than the male controls. (for details see Table 1)
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
There was a slight but consistent reduction in food consumption by 5% adipic acid.
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
When surviving animals were sacrificed at the end of the two-year period, there was no significant gross pathology that could be related to ingestion of the compound. There was an equal incidence of mottled, granular livers with peripheral thickening in both the control  and experimental animals. 
The results of microscopic examination appeared to be within normal limits for the representative tissues.
Histopathological findings: neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Two of the surviving control animals and one of the experimental animals had ovarian tumors, ovarian cysts were noted in both control and experimental rats.
Dose descriptor:
NOAEL
Effect level:
ca. 750 mg/kg bw/day (actual dose received)
Based on:
test mat.
Remarks:
(1% in diet)
Sex:
male/female
Basis for effect level:
body weight and weight gain
Critical effects observed:
not specified

In the course of the study body weights were affected in males at 3% and 5% adipic acid in the diet (see Table 1). In the high dose group males a consistently lower body weight was recorded throughout the study, reaching 32% lower body weights than control males 8 weeks after start of treatment. At the end of the study the body weight of males was reduced by 9% in the 3% dose group and by 18% in the 5% dose group.

Table 1: body weight development in g (percent difference to respective control animals)

 week  control males  0.1% males  1% males  3% males  5% males  control females  1% females
 0  59  61  63  61  57  49  48
 8  269  280  265  224 (-17%)  182 (-32%)  178  175
 16  325  333  320  276 (-15%)  233 (-28%)  222  213
 24  361  374  354  309 (-14%)  264 (-27%)  242  233
 32  377  391  376  329 (-13%)  291 (-23%)  257  249
 ...              
 64  426  455  436  385 (-10%)  339 (-20%)  295  284
 ...              
 104  440  417  437  400 (-9%)  360 (-18%)  321  304


Table 2: food and test material consumption, survival

 level  sex  food consumed (g average/rat/day)  compound consumed (mg average/rat/day)  survival
 control  male  16.8  -  82.5%
   female  14.2  -  98.9
 0.1% adipic acid  male  17.0  17.0  87.7
 1% adipic acid  male  17.5  175  94.7
   female  15.8  158  96.3
 3% adipic acid  male  16.8  505  94.5
 5% adipic acid  male  15.7  814  97.2


There was no evidence of gross pathology associated with the feeding of adipic acid. There was no significant difference in survival. The results of microscopic examination were to be within normal limits.

Autopsy data for the male animals that died during the course of the two-year feeding program and for the sacrificed rats were analyzed for incidence of tumors and/or lung pathology. The incidence of lung pathology, tumors, soft testes observed in the adipic acid treated groups was as frequent as in the control group.

Female animals, dosed with 1% adipic acid and controls, exhibited signs normally associated with advancing senility in rats in the last six months. There was an equal incidence of blood-tinged crust about the eyes and noses,
unthriftiness, and body scores in both groups. A few control and experimental animals had alopecia, and one experimental
rat appeared to develop a middle ear infection during the 102nd week. One experimental and two control animals died
during the final six months. All three exhibited diarrhea, respiratory infection and loss of body weight prior to
death. Upon autopsy, one control rat and one experimental rat were found to have tumors, while the other control
animal had a granular liver and dark red apexes on both lungs. 

Comparison of the chronic feeding of acipic acid with citric acid (3% and 5% in the diet, also reported in this paper) indicates that adipic acid is comparable with citric acid.

Executive summary:

In a two-year study, groups of 20 male rats were given 0.1, 1, 3 and 5 % of adipic acid in the diet (equivalent to doses of approximately 75, 750, 2250 and 3750 mg/kg bw/day). Groups of 19 or 10 female rats received food containing 1 % adipic acid (approx. 750 mg/kg bw/day) or plain diet, respectively. Body weights, food consumption and general appearance were recorded weekly throughout the experimental period. After 2 years, surviving rats were weighed, killed, and examined grossly. The brain, thyroid, lung, heart, liver, spleen, kidneys, adrenals and stomach of the animals were weighed. Microscopic examination of thyroid, lung, heart, liver, spleen, kidneys, adrenals, stomach, pancreas, bone marrow, large and small intestine uterus, ovaries and testes on a representative number of animals (no further information) was performed.

The percent survival for each test group was not negatively affected by treatment. There were no body weight differences during the test period in female and male rats treated with 0, 0.1 and 1 % adipic acid. The body weights of the male rats receiving 3 and 5 % adipic acid were distinctly lower than the control group. In the 5% males up to minus 32% body weight were recorded. At necropsy there was no treatment related effect on organ weights observed. Results of microscopic examination of the organs revealed no compound related effect. The NOAEL was 1 % for male and female rats (approx. 750 mg/kg bw/day) (Horn et al. 1957). The study does not fully comply with the guidelines for chronic studies because microscopic examination was limited to 15 tissues of a representative number of animals of each group, females received only one dose, the MTD was reached only for males, and the purity of adipic acid is not indicated.

Data source

Reference
Reference Type:
publication
Title:
Safety of adipic acid as compared with citric and tartaric acid.
Author:
Horn HJ, Holland EG, Hazleton LW
Year:
1957
Bibliographic source:
Agricult. Food Chem. 5, 759-762.

Materials and methods

Principles of method if other than guideline:
see repeated dose toxicity Horn et al. 1957. Rats were fed either the basal laboratory diet, or the basal diet to which adipic acid was added. Body weights, food consumption, and general appearance were recorded weekly throughout the experimental period. Whenever possible, gross autopsy was performed on those animals that died during the course of the experiment. After two years, surviving rat were weighed, killed, and examined grossly. Organs were weighed. Microscopic examination of several organs, including testis or ovaries and uterus was performed on a representative number of animals.
GLP compliance:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Adipic acid
EC Number:
204-673-3
EC Name:
Adipic acid
Cas Number:
124-04-9
Molecular formula:
C6H10O4
IUPAC Name:
adipic acid
Details on test material:
Test substance purity not specified

Test animals

Species:
rat
Strain:
other: Carworth Farm strain
Sex:
male/female
Details on test animals or test system and environmental conditions:
weight at study initiation: 50-60 g
housing individually in cages with wire mesh floors
free access to food and water

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on mating procedure:
no mating
Duration of treatment / exposure:
Exposure period: 2 years
Frequency of treatment:
diet ad libitum
Details on study schedule:
2 year feeding study
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations for males:
0.1, 1, 3, and 5%; (approx. 75, 750, 2250, 3750 mg/kg bw/day)
Basis:
nominal in diet
Remarks:
Doses / Concentrations in females
1%; (ca. 750 mg/kg bw/day)
Basis:
nominal in diet
No. of animals per sex per dose:
started with 20 males per treatment groups and in the control group
started with 19 females in the treatment group and 10 females in the control group
Control animals:
yes, plain diet
Positive control:
not applicable

Examinations

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

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- 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

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
The following signs were observed among all male groups, including the controls, especially during te final six months: wheezing, blood-tinged crust about the noses and eyes, and body sores. These findings were not significantly different among the groups, although a lower incidence of signs indicative of respiratory infection and body sores occurred in the 5% adipic acid group.
Description (incidence):
The per cent survival was not negatively affected by treatment.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Growth of males treated with 0.1 and 1% and females treated with 1% test item in food was comparable to that of the respective controls. During the rapid growth period of the 2-year feeding studies, weight gains for the male rats receiving 3 or 5% adipic acid was significantly less (up to minus > 30%) than the male controls.
(for details see Table 1 in IUCLID chapter 7.5.1)
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
There was a slight but consistent reduction in food consumption by 5% adipic acid.
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Microscopic examination of the following reproductive organs was performed: uterus, ovaries and testes on a representative number of animals (no further information)
When surviving animals were sacrificed at the end of the two-year period, there was no significant gross pathology that could be related to ingestion of the compound. The results of microscopic examination appeared to be within normal limits for the representative tissues.
Two of the surviving control animals and one of the experimental animals had ovarian tumors, ovarian cysts were noted in both control and experimental rats.

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
ca. 750 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
Remarks on result:
other: according to 1% in the diet

Target system / organ toxicity (P0)

Critical effects observed:
no

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Males (control, 0.1, 1, 3, 5% adipic acid; 20 male animals/group): When the surviving males were sacrificed there was no significant gross pathology that could be related to adipic acid. Histopathologic examination of the testes revealed no evidence of an adverse effect on the reproductive organs up to the highest dose. Soft edematous testes were noted at least as frequent in the controls as in the experimental animals.  Females (10 control animals and 19 animals dosed with 1% adipic acid): When the surviving females were sacrificed there was no significant gross pathology that could be related to adipic acid. Histopathologic examination of the ovaries and uterus revealed no evidence of an adverse effect on the reproductive organs. Two of the surviving control animals and one of the experimental animals had ovarian tumors, ovarian cysts were noted in both control and experimental rats.

In summary: histopathologic examination of the testes, ovaries and uterus revealed no evidence of an adverse effect on the reproductive organs.

Applicant's summary and conclusion

Executive summary:

Studies on fertility are not available. In a two-year feeding study in rats (see IUCLID chapter 7.5.1 Repeated Dose Toxicity) histopathological examination of testes, ovaries and uterus revealed no evidence of an adverse effect on the reproductive organs up to the highest tested doses (3750 mg/kg bw/day in males, 750 mg/kg bw/day in females). The NOAEL for maternal toxicity was about 750 mg/bw/day, based on body weight effects at higher doses in males. The NOAEL for effects on reproductive organs was the highest dose tested (3750 mg/kg bw/day in males, 750 mg/kg bw/day in females).

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