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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
Study period:
3 July 1994 to 7 August 1994
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, equivalent to a valid guidelines and the study was conducted under GLP conditions. The study was performed with test material being used to support the substance on the basis of read-across.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1995
Report Date:
1995

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
liquid
Details on test material:
- Name of test material (as cited in study report): Cyclohexylsalicylate (abbreviated as CHS)
- Physical state: colourless liquid
- Storage condition of test material: room temperature
- Molecular formula (if other than submission substance): C13H16O3
- Molecular weight (if other than submission substance): 220.27
- Smiles notation (if other than submission substance): C(=O)(c1c(O)cccc1)OC1CCCCC1
- Stability under test conditions: Stable in arachidis oil, DAB 10

Test animals

Species:
rat
Strain:
other: Hsd/Win:Wu
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Winkelmann, D-33178, Borchen
- Age at study initiation: approximately 5 weeks
- Housing: Makrolon Type M 5 cage (housed in pairs or in threes)
- Diet: Pelleted Altromin Maintenance Diet 1324 ad libitum
- Water: tap water ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature: 19-24°C
- Humidity: 39-65%
- Air changes: 10-15 air changes per hour
- Photoperiod: 12 hrs dark / 12 hrs light

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: archadis oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test doses were formulated every day prior to administration. Animals were dosed within 2-3 hours of dose preparation.
The dose volume applied was 5 mL/kg bw.

40 mg/kg bw/day: concentration in vehicle 8.0 mg/mL
120 mg/kg bw/day: concentration in vehicle 24.0 mg/mL
360 mg/kg bw/day: concentration in vehicle 72.0 mg/mL

Control animals were dosed with the vehicle only at a dose volume of 5 mL/kg bw.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The solution was analysed once for achieved concentration using HPLC.
Duration of treatment / exposure:
90 days (with an additional 28 days for the recovery groups)
Frequency of treatment:
Daily, five days per week. Animals were given a total of 68/69 applications of the test material.
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
control
Dose / conc.:
40 mg/kg bw/day (nominal)
Dose / conc.:
120 mg/kg bw/day (nominal)
Dose / conc.:
360 mg/kg bw/day (nominal)
No. of animals per sex per dose:
Ten (five for the recovery groups)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The rationale for dose levels based on the results of toxicological examinations (reports cited in study report).


- Post-exposure recovery period in satellite groups: An additional five males and five females were included in each of the 0 and 360 mg/kg bw test groups. After the last exposure, the animals were allowed 29 days recovery.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: treatment period - twice daily (once daily weekends and public holidays), treatment-free period - once daily
- Cage side observations checked: mortality and morbidity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: treatment period - twice daily (once daily weekends and public holidays), treatment-free period - once daily
- Cage side observations checked: mortality and morbidity

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION: yes
- Time schedule for examinations: weekly (recorded per cage)

WATER CONSUMPTION: yes
- Time schedule for examinations: weekly (recorded per cage)

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: start and end of treatment.
- Dose groups that were examined: all

HAEMATOLOGY: Yes
- Time schedule for collection of blood: after six weeks of administration (intermediate analysis) and at the end of analysis
- Anaesthetic used for blood collection: Yes (ether)
- Animals fasted: Yes
- How many animals: all
- Parameters checked: red blood cell count, haematocrit, mean corpuscular volume, haemoglobin, white blood cell count, platelet count and differential white blood cell count (bands, polymorphnuclear neutrophils, lymphocytes, polymorphnuclear eosinophils, monocytes, polymorphnuclear basophils, myelocytes, juvenil, diverse)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: after six weeks of administration (intermediate analysis) and at the end of analysis
- Animals fasted: No data
- How many animals: all
- Parameters checked: Liver profile: gamma-glutamyl transferase (GGT), aspartate-amino-transferase (AST), alanine-amino-transferase (ALT) and bilirubin. Adrenals profile: sodium, potassium, glucose. Kidney profile: urea, sodium and potassium. Mineral metabolism and parathyroid gland profile: Calcium and alkaline phosphatase. Electrolytes and water balance profile: Sodium, potassium, chloride and total protein. Lipid metabolism profile: total cholesterol.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
- Parameters checked: relative and absolute organ weights (brain, testes, heart, liver, spleen, adrenals, kidneys and thymus)

HISTOPATHOLOGY: Yes
- Parameters checked: adrenals, aorta, bone marrow, brain, caecum, colon, duodenum, epididymis, eyes, heart, ileum, jejunum, liver, lungs, lymph node, (submandiburalis, mesenteric, inguinalis), mammary gland, oesophasgus, ovaries, pancreas, parathyroids, prostate, rectum, salivary glands, skeletal muscle, skin, spinal cord, spleen, stomach, testes, thymus, thyroids, trachea, urinary bladder, uterus, lumbal vertebrate and all gross lesions.
Statistics:
t-test for determination of significant differences between the groups for body weight gains. t-test for the determination of significant differences between the groups for haematological and clinical chemistry examinations. Steel-test for the determination of significant differences between the groups for organ weight gains.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, non-treatment-related
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
None of the treated animals showed any mortality or clinical effects. A small number of animals showed slight to moderate acute swelling of the right ear forming a haematoma. The swelling was not histologically examined because no treatment-relationship was found in an earlier experiment and was assumed to be related to the ear tattoo used to identify the animals.

BODY WEIGHT AND WEIGHT GAIN
The body weight gain of group 2 females, group 3 males and females and group 4 females was similar to the controls. The group 2 males showed a slight decrease in body weight gain in weeks 10, 11 and 13 and the males in group 4 showed a slight decrease in week 1. The slight decrease in group 4 males is because four animals had a low initial body weight in comparison to the controls and is considered to not be treatment-related (see Table 1).

FOOD CONSUMPTION
The mean food consumption of the treated animals showed some deviations which were within the normal range of historical values and so were considered not not be treatment-related.

FOOD EFFICIENCY
The mean food conversion was comparable to the control groups (see Table 2).

WATER CONSUMPTION
The mean water consumption of the animals in groups 2 and 3 sometimes showed distinct but no dose-related deviations which were not considered to be treatment-related. The group 4 males showed an increase from week 3 to week 13 and the group 4 females showed an increase over the whole study. The mean water conversion was comparable to the control groups.

OPHTHALMOSCOPIC EXAMINATION
No treatment-related effects were observed.

HAEMATOLOGY
Intermediate analysis: in the treated groups, no significant deviations were observed in any parameter and all values were similar to the controls.

Final analysis: in groups 2 and 3 and group 4 males, no significant deviations were observed in any parameter and all values were similar to the controls. In group 4 females, a slight increase of the polymorphnuclear neutrophils in the differential white blood count was recorded. This significant finding was considered to be within the normal range of historical values.

CLINICAL CHEMISTRY
Intermediate analysis: a slight increase in chloride in males in groups 2 and 3, an increase in chloride, sodium and creatinine and decrease in bilirubin in group 4 males and an increase in chloride and sodium and a decrease in bilirubin in group 4 females was observed.

Final analysis: in groups 2 and 3 and group 4 females, no significant deviations were observed in any parameter and the values were similar to the controls. A slight decrease in bilirubin was observed in group 4 males; these significant findings were considered as within the normal range of historical values.

ORGAN WEIGHTS
A slight decrease in the absolute liver weights of group 3 males and a slight increase in the absolute liver weights of group 3 females were observed.

A slight increase in relative brain weights in group 2 males and a slight increase in relative liver weights in group 4 females were observed. The slight increase in the relative liver weights of the group 4 females was not dose-dependent and therefore the increase was considered to be incidental.

GROSS PATHOLOGY
Various spontaneous findings in males and females of all groups such as hydrometra of the uterus, foci of the liver, hydronephrosis and other diagnoses were observed. Treatment-related findings were not observed in the treated animals. The recovery groups did not show macroscopical substance-related findings.

HISTOPATHOLOGY
Findings included spontaneous lesions in males and females of all groups such as hyperlasia of the mammary gland, hydrometriosis of the uterus, accumulations of lymphocytes in the lungs and other spontaneous lesions.

The liver of four group 4 males showed small areas of necrosis with a chronic character. In order to determine whether these were treatment-related, all livers of all animals in groups 1 to 4 and the recovery groups were examined. No other necrotic foci were observed in the livers of the group 4 males and females. Small areas of infarction were observed in the males of groups 1 to 3 but no infarction was observed in any females. No further developments e.g. acute lesions within the livers were observed and therefore the chronic necrotic areas were considered as incidental and not substance-related. Furthermore, one animal in the control group showed a similar necrotic area in the liver.

Effect levels

Dose descriptor:
NOEL
Effect level:
360 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Daily administration at dose levels of 40 up to 360 mg/kg bodyweight revealed no systemic or toxic effects in all main test groups

Target system / organ toxicity

Critical effects observed:
no

Any other information on results incl. tables

Table 1: Body weights and weight gains in group mean values (g)

 Group, sex
 Week  1, m  2, m  3, m  4, m  1, f  2, f  3, f  4, f
 1  150  148  149  143*  124  122  124  124
 2  198  195  197  191  150  150  153  149
 3  247  241  242  239  138  169  172  168
 4  284  275  278  276  185  185  190  185
 5  313  302  306  304  196  199  202  197
 6  340  330  333  331  208  211  214  208
 7  363  349  353  353  214  220  223  218
 8  375  360  365  364  219  224  227  221
 9  395  376  380  382  225  232  232  228
 10  408  385*  390  393  231  238  236  232
 11  420  397*  404  409  234  244  239  237
 12  428  406  416  419  241  248  243  240
 13  437  412*  419  426  241  252  248  244
 14  444  420  426  431  245  253  249  245
 Gain (1 - 7)  214  201  203  209  89  98  99  94
 Gain (7 - 14)  81  72  73  78  31  34  26  27
 Gain (1 - 14)  295  273  276  287  120  131  125  121

* 95% significance in comaprison with the controls

Table 2: Food consumption group mean values (g/rat/week)

 Group, sex
 Week  1, m  2, m  3, m  4, m  1, f  2, f  3, f  4, f
 1  157  150  153  151  119  121  120  119
 2  158  152  151  157  117  118  119  121
 3  168  154  155  160  106  109  111  110
 4  169  152  152  166  115  118  121  121
 5  169  154  158  169  116  115  116  122
 6  174  161  169  177  124  122  121  125
 7  -  -  -  -  -  -  -  -
 8  159  145  145  158  109  107  107  115
 9  173  173  162  171  116  120  116  120
 10  166  150  150  165  114  121  116  116
 11  159  148  152  169  114  115  111  111
 12  157  143  145  164  117  117  114  112
 13  154  140  135  155  116  108  108  111
 Mean 1 - 13  163  152  152  163  115  116  115  117

Table 3: Water consumption group mean values (g/rat/week)

 Group, sex
 Week  1, m  2, m  3, m  4, m  1, f  2, f  3, f  4, f
 1  198  195  196  198  170  172  163  182
 2  233  221  214  217  167  165  167  181
 3  231  228  227  248  160  173  184  194
 4  233  238  220  257  164  168  180  192
 5  236  238  222  260  169  175  182  198
 6  220  254  237  261  176  179  169  205
 7  -  -  -  -  -  -  -  -
 8  223  241  207  269  161  181  174  193
 9  254  252  237  281  182  188  175  212
 10  238  244  221  263  180  191  188  196
 11  229  235  237  274  172  180  172  198
 12  243  242  219  281  191  191  184  210
 13  242  263  244  272  186  207  193  200
 Mean 1 - 13  232  238  223  257  173  181  178  197

Applicant's summary and conclusion

Conclusions:
Under the conditions of the test, the NOEL was determined to be 360 mg/kg bw/day. Daily administration of the test material at 40 to 360 mg/kg bw revealed no systemic or toxic effects in test groups 2 to 4. Food consumption, body weight gain, haematology and clinical chemistry were in the normal range and revealed no treatment-related effects. Water consumption in group 4 males and females was at a higher level than that observed in the other test groups. The higher water consumption caused no related lesions in group 4 animals. The results of the intermediate analysis of the clinical chemistry showed some increases/decreases in various parameters (chloride, bilirubin, sodium); in the final analysis only a slight decrease in bilirubin in group 4 males was seen but was considered to be incidental. All animals of the other groups showed no significant deviations. A slight increase in absolute and relative weights of group 3 and 4 females was seen along with a slight decrease in absolute liver weights in group 3 males; these findings were considered to be incidental.

Four livers (out of 10) in group 4 males showed various necrotic areas of chronic character. Beside the change in body weight gain in these animals, no other findings could be evaluated including those related to liver metabolism. One animal in each of groups 1 to 3 showed a similar alteration and the presence of the necrotic areas was therefore considered to be incidental. No lesions were observed in all other group 2 and 3 and recovery group animals.
Executive summary:

The objective of the study was to determine the toxicity of the test material following an oral (gavage) administration to rats for 90 days and evaluate the possible reversibility of any toxic signs following a 29 day treatment-free period. The test material was tested in doses of 0 (group 1), 40 (group 2), 120 (group 3) and 360 (group 4) mg/kg bw with ten males and ten females used for each dose group. In addition, five males and five females were added to groups 1 and 4 and were allowed to recover for 29 days after the last administration.

There were no unscheduled deaths. There were no treatment-related difference in body weight gain. Both the food consumption and food conversion of the different groups were similar. There were treatment-related differences in the amount of water consumption of the group 4 animals compared to the controls; there was no differences in the water conversion between different groups. The hematological and clinical chemistry studies showed no biological significant differences between groups 1 and groups 2 - 4. There were no eye lesions observed by slit lamp ophthalmology. There were no treatment-related differences in the absolute and relative organ weights. There were no treatment-related macroscopical or histological lesions at necropsy at the end of the treatment or treatment-free period.

The test material is considered sufficiently similar to (z)-3-hexenyl salicylate on the basis of read-across such that the results from this study can be used to address the acute oral toxicity of (z)-3-hexenyl salicylate.