Registration Dossier

Administrative data

Description of key information

Repeated dose toxicity:
- 90-d oral toxicity study: NOAEL ≥ 150 mg/kg bw/day

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
April - August 1995
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Test was performed according to OECD TG 408 and under GLP. Highest dose level did not cause a significant toxic response of the test animals.
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
yes
Remarks:
highest dose did not cause a toxic effect.
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Crl:CD (SD) Br
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Limited, Margate, England
- Age at study initiation: 43-46 days old
- Weight at study initiation: males: 182 - 266 g; females: 151-201 g
- Fasting period before study: no
- Housing: The animals were housed singly, in grid-bottomed cages, suspended over cardboard-Imect excreta trays.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 18 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): The experimental room (W9) was air conditioned and recorded temperature was generally in the target range of 19 – 25 degrees C. The temperature dropped below the target range on five occasions (16 degrees C or 18 degrees C and exceeded it once (27 degrees C).
- Humidity (%): Relative humidity was generally in the target range of 30 - 70%. The humidity exceeded the target range on six occasions (up to 83%).
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light): Fluorescent lighting was controlled automatically to give a cycle of 12 hours light (0600 to 1800 hours) and 12 hours dark.


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


DIET PREPARATION
All animals were offered a powdered diet (SQC Rat and Mouse Maintenance Diet No. 1, Expanded, Ground Fine, Special Diets Services Limited, Witham, England)

Each batch of diet was delivered with an accompanying certificate of analysis detailing nutritional composition and levels of specified contaminants (heavy metals, aflatoxins and insecticides). The water was periodically analysed for microbiological purity and levels of metals and halogenated hydrocarbons by independent analysts (Acer Environmental, Bridgend, U.K.).

The criteria for acceptable level of contaminants in stock diet and water supply were the analytical specifications established by the diet manufacturer and local water authority respectively. It was considered that none of the contaminants that were monitored in the diet and water was likely to be present at a level which would have prevented the study objectives from being achieved.

Test diets were prepared freshly each week, according to known stability. A weighed amount of test article was first dissolved in an appropriate amount of acetone. This was then added to a small quantity of untreated diet, then mixed using a Kenwood Chef static mixer. Further quantitites of untreated diet were then added until a premix of typically 1kg was obtained. For each group, the appropriate quantity of premix was then added to the required quantity of untreated diet to obtain the desired batch size, then mixed for at least 15 minutes in a double cone rotary mixer.
For the control diet, a quantity of acetone similar to that used during preparation of the diet for the high dose group was added during preparation.

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of the diets for each group (including that for the control group) prepared for use during weeks 1, 7 and 13 were analysed for test article concentration by Toxicol Laboratories using a previously validated method. On each occasion, the prepared diets were analysed for residual acetone.
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
daily, 7 days a week
Remarks:
Doses / Concentrations:
5, 15, 50, 150 mg/kg bw/day
Basis:
nominal in diet
No. of animals per sex per dose:
15 males and 15 females per dose group
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale:
Doses for this study were selected based on the results of a 2 week oral (dietary) rangefinding and palatability study in the rat performed at Toxicol Laboratories (Study Number RFAl4/95). The concentrations (ppm) of HHCB in the test diets were adjusted weekly in order to achieve a constant target dose based on mg/kg bodyweightlday. The doses are in terms of nominal doses.
- Rationale for animal assignment (if not random):
Fifteen days before the start of treatment, the required number of animals was obtained by weighing all animals and discarding those at the extreme of the weight range. Selected animals were then allocated randomly to groups. Cage positions on the battery were allocated by starting at the top lef hand corner of the battery and working left to right and top to bottom placing a cage from each group of males in ascending group order. The sequ nce was repeated until all the males were allocated, and was then performed similarly for females.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
All animals were examined daily for any visible signs of reaction to treatment



BODY WEIGHT: Yes
- Time schedule for examinations: All animals were weighed once during acclimatization, a second time 1 day prior to treatment and weekly thereafter.



FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
Food consumed by each animal was recorded over 6 days in each week
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes


OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations:
- Dose groups that were examined:
All animals were subjected to ophthalmologic examination before the start of the treatment. Animals in the control and high dose groups were also examined during week 13. Those animals selected for the treatment-free period were examined at the end of that period.



HAEMATOLOGY: Yes
- Time schedule for collection of blood: conducted for all animals once each during weeks 7 and 13 of the treatment period and at the end of the treatment-free period
- Anaesthetic used for blood collection: No data
- Animals fasted: No
- How many animals: all
- Parameters examined: hemoglobin concentration, red blood cell count, packed cell volume, mean cell volume, mean cell hemoglobin,
mean cell hemoglobin concentration, total leukocyte count, platelet count and leukocyte differential count



CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: conducted for all animals once each during weeks 7 and 13 of the treatment period and at the end of the treatment-free period
- Animals fasted: No
- How many animals: all
- Parameters examined: albumin/globulin ratio, alanine aminotransferase, albumin, alkaline phosphatase, aspartate amino-transferase, blood urea nitrogen, calcium, chloride, cholesterol, creatinine, gammaglutamyltransferase, glucose, inorganic phosphorous, potassium, sodium, bilirubin, total protein, triglycerides



URINALYSIS: Yes
- Time schedule for collection of urine:
Overnight urine samples were collected from all animals under food and water deprivation once during weeks 6 and 12 of the treatment period and at the end of the treatment-free period
- Metabolism cages used for collection of urine: No data
- Animals fasted: Yes
- Parameters examined: bilirubin, blood pigments, glucose, ketones, leukocytes, nitrites, pH, protein and urobilinogen. In addition, volume, color, appearance and specific gravity were recorded and there was a microscopic examination of sedimented deposits.



Sacrifice and pathology:
GROSS PATHOLOGY: Yes
Organ weights ( brain, heart, kidneys, liver, lungs, ovaries, pituitary, spleen, testes, thymus and thyroids) were recorded

HISTOPATHOLOGY: Yes
brain, heart, kidneys, liver, lungs, ovaries, pituitary, spleen, testes, thymus and thyroids, aorta, brain (three levels), cecum, colon, duodenum, epididymides, eyes (including optic nerve), femur (including marrow), ileum, jejunum, lachrymal glands (exorbital), mainstem bronchi, mammary gland, mesenteric lymph node, esophagus, pancreas, prostate, rectum, salivary gland maxillary and sublingual), sciatic nerve, seminal vesicles, skin, spinal cord (three levels), sternum (including marrow), stomach, submandibular lymph node, trachea, urinary bladder, uterus and vagina. All tissues from the high dose groups and controls as well as any others showing any grossly observable effects were examined microscopically.
Statistics:
The data were subjected to an analysis of variance (ANOVA). Males and females were analyzed separately. Levene’s test was performed to determine if there were any between group differences in variances. If Levene’s test indicated that there was no evidence of inequality of variance, then pairwise tests of all treated groups versus control were performed using Williams’ test. For the comparison of the high dose against the control, a two-sided test was performed. Statistical significance was declared at the two-sided 5% level and also noted at the 1 and 0.1% levels. If Levene’s test indicated that there was evidence of differences between group variances or if the data were judged to be unsuitable for a parametric analysis, then a non-parametric approach to the data analysis was used. This involved the Kruskal -Wallis non-parametric ANOVA and Shirley’s non-parametric equivalent of Williams’ test.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY


BODY WEIGHT AND WEIGHT GAIN
Bodyweight gain was increased in males, but not females, at all doses. This increase was not statistically significant and no correlation with dose was seen.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
Food consumption was increased in males, but not females, at all doses. This increase was not statistically significant and no correlation with dose was seen


OPHTHALMOSCOPIC EXAMINATION
No changes in ophthalmologic evaluation were observed

HAEMATOLOGY
No changes in hematology were observed

A statistically significant higher mean partial thromboplastin time (PTT) was seen in the females at 150 mg/kg bw/day (16.6 sec.) and 50 mg/kg bw/day (16.5 sec.) compared to controls (14.3 sec) at week 13. These values were slightly higher than the high end of the historical controls. The increase in PTT can indicate acquired hypofibrinogenemia. However, in this study, the abnormal height of control fibrinogen might explain the increase in PTT. Moreover, there is no clear dose-response relationship, a lack of other corresponding findings such as effects on liver cells (absence of histomorphological alterations, absence of liver enzyme activation), a lack of any other indication of abnormal (retarded) coagulation and no signs of uncontrolled spontaneous bleeding such as abnormal occurrence of petechia or hemorrhagia in organs, skin or mucous membranes. There were no significant differences in prothrombin times in any of the groups (males and females) in comparison with their respective controls. All groups of treated females had statistically significant lower fibrinogen than controls, without a clear dose-response, while the mean fibrinogen level for the female control group was significantly higher than the highest historical control value. At the end of the treatment-free period, no significant effect was found on either fibrinogen, partial thromboplastin time or prothrombin time in females nor males. For these reasons, the deviations in PTT are not considered of toxicological relevance.

The mean red blood cell counts in males but not females at week 7 in the 150 and 50 mg/kg bw/day groups were significantly slightly lower than in the controls. At week 13, the males did not differ significantly from the controls in any group however the high dose females were slightly lower than controls.
The total leucocyte count was significantly lower for the females in the 15, 50 and 150 mg/kg bw/day groups compared to controls at week 7, without any dose-response relationship.
Statistically significant lower monocytes were reported for males, but not females, at all but the low dose group at week 13 but not 7. The imprecision of the reported values makes significance questionable.
The packed cell volumes for males at 50 and 150 mg/kg bw/day were statistically significantly lower than controls at week 7 but not week 13. There was no correlation with dose. The mean haemoglobin concentrations for all dosed groups at week 13, all dosed females at week 7 and for the two highest dosed male groups at week 7 were statistically significantly higher than the respective controls but with no correlation with dose and all values, except for that of the males at 50 mg/kg bw/day at week 7, were within the historical control ranges.
ALT levels were statistically significantly lower in females but not males in the two higher dosed groups at week 7. They were not significantly different in males or females at week 13. AST levels were statistically significantly lower than controls in males at all dose levels but only in the highest dosed females at week 7. However, at week 13, all but the lowest dosed females had statistically significantly lower AST levels with no significant differences in any male group.


CLINICAL CHEMISTRY
At weeks 7 and 13, slightly lower mean plasma triglyceride levels were observed for males given 15, 50 and 150 mg/kg per day. A slightly lower plasma glucose concentration was noted at week 7 in males and females given 15, 50 and 150 mg/kg per day, and at week 13 in males given 50 and 150 mg/kg per day. However, all mean values, except for the high dose males at week 13, were within the standard deviation of the controls. None of these differences between control and treated groups were present at the end of the treatment-free period.


URINALYSIS
During weeks 6 and 12, a faint/light brown discoloration of the urine was observed for a small proportion of males given 50 or 150 mg/kg per day, but this was not evident at the end of the treatment-free period.


ORGAN WEIGHTS
There were no statistically significant differences in absolute or bodyweight related organ weights after 13 weeks of treatment. However, when liver weight was compared with brain weight, statistically significant higher values were seen in all dosed males compared to control males. There was no evidence of a dose response relationship


GROSS PATHOLOGY
No abnormalities of any tissues were observed at necropsy

HISTOPATHOLOGY: NON-NEOPLASTIC
No abnormalities of any tissues were observed in histopathology. Distension of the uterine lumen was observed in all dose groups, with slightly increased incidences in the groups treated with 5 and 150 mg/kg per day but no significant difference in the 50 mg/kg per day group and a decreased incidence in the 15 mg/kg per day group compared with controls Histopathological examination of the ovaries and vaginas showed an association between the distension and the proestrus stage of the estrus cycle. No other histopathological effects in male or female reproductive organs were found in any dose group.


HISTOPATHOLOGY: NEOPLASTIC (if applicable)


HISTORICAL CONTROL DATA (if applicable)


OTHER FINDINGS
Dose descriptor:
NOAEL
Effect level:
150 other: mg/kg
Sex:
male/female
Critical effects observed:
not specified

Table 1. A Selected haematological parameters for HHCB (n=15/group)

Dose (mg/kg bw/day)

Partial thromboplastin time (sec)

Partial thromboplastin time (sec)

Fibrinogen (mg/dl)

Fibrinogen (mg/dl)

RBC

RBC

RBC

RBC

WBC

WBC

WBC

WBC

Male W13

Female W13

Male W13

Female W13

Male  W7

Female W7

Male W13

Female W13

Male W7

Female W7

Male W13

Female W13

0

17.0 ± 1.46

14.3 ± 1.72

303 ± 39.5

302 ± 68.2

8.7 ± 0.30

8.2 ± 0.34

9.0 ± 0.35

8.6 ± 0.34

14.6 ± 4.10

12.6 ± 3.14

13.0 ± 2.74

8.5 ± 1.23

5

17.9 ± 2.02

15.8 ± 3.93

317 ± 54.9

250 ± 40.6b

8.5 ± 0.30

8.1 ± 0.29

8.8 ± 0.43

8.4 ± 0.30

15.4 ± 3.11

11.2 ± 3.13

14.3 ± 3.12

8.1 ± 3.62

15

16.2 ± 1.63

15.5 ± 1.75

301 ± 52.1

253 ± 29.0b

8.5 ± 0.29

8.2 ± 0.32

8.9 ± 0.38

8.5 ± 0.44

15.4 ± 3.97

9.5 ± 2.74b

12.6 ± 2.88

6.7 ± 1.42

50

17.8 ± 2.78

16.5 ± 2.65a

307 ± 25.3

250 ± 38.2b

8.3 ± 0.33a

8.1 ± 0.28

8.9 ± 0.32

8.4 ± 0.24

14.3 ± 3.39

8.5 ± 3.05b

12.6 ± 3.07

7.6 ± 2.41

150

17.9 ± 3.52

16.6 ± 2.28a

298 ± 40.9

241 ± 41.5b

8.4 ± 0.35a

8.3 ± 0.31

8.8 ± 0.36

8.3 ± 0.31a

15.2 ± 5.29

10.0 ± 3.76b

12.4 ± 3.52

7.8 ± 3.14

HH

19.7

16

394

285

8.9

8.6

9.8

9.2

18.0

15.0

21.0

14.6

HM

15.1

12.6

292

234

7.7

7.6

9.0

8.4

12.7

10.0

14.2 ±

9.5

HL

10.5

9.2

191

182

6.5

6.6

8.2

7.6

7.4

5.0

7.4

4.4

Mono

Mono

Mono

Mono

PCV

PCV

PCV

PCV

MCHC

MCHC

MCHC

MCHC

Male W7

Female W7

Male W13

Female W13

Male W7

Female W7

Male W13

Female W13

Male W7

Female W7

Male W13

Female W13

0

2 ± 1.2

2 ± 1.6

4 ± 1.5

2 ± 1.9

45.2 ± 1.31

44.8 ± 1.98

46.1 ± 1.40

46.4 ± 2.01

36.9 ± 0.52

36.5 ± 1.05

36.2 ± 0.57

36.2 ± 0.50

5

3 ± 1.8

2 ± 1.4

4 ± 1.7

1 ± 1.1

44.6 ± 1.10

43.5 ± 1.36

45.5 ± 1.79

45.1 ± 1.88

37.0 ± 0.39

37.6 ±0.50c

36.6 ± 0.55a

36.7 ± 0.58a

15

2 ± 1.1

2 ± 1.4

2 ± 2.0a

2 ± 1.5

44.7 ± 1.15

43.5 ± 1.07

46.2 ± 1.70

45.2 ± 1.64

37.0 ± 0.77

37.3 ±0.77c

36.4 ± 0.61a

36.6 ± 0.73a

50

3 ± 1.5

2 ± 2.1

2 ± 1.8b

2 ± 1.6

43.5 ± 1.36b

43.7 ± 1.24

45.0 ± 1.52

45.6 ± 1.01

37.7 ± 0.46b

37.1 ±0.46c

36.9 ± 0.46c

36.8 ± 0.52b

150

2 ± 1.4

2 ± 1.7

3 ± 1.7a

1 ± 1.1

44.1 ± 1.49b

44.3 ± 1.36

45.2 ± 1.64

45.2 ± 1.59

37.3 ± 0.53b

37.4 ±0.54b

36.9 ± 0.37c

36.9 ± 0.45b

HH

5

5

5

4

49.7

48.1

50.7

49.3

37.5

37.9

37.4

37.5

HM

2

2

2

1

44.5

43.7

46.9

45.2

35.1

35.6

35.2

35.3

HL

0

0

0

0

39.3

39.3

43.1

41.1

32.7

33.3

33.0

33.1

M: male rats; F: female rats; W7: week 7 observations; W13: week 13 observations                            HH: historical high values; HM: historical mean values; HL: historical low values

(for measurements at W7, values from historical animals 0-3 months of age are used and for measurements at W13, values from animals 4-6 months are used.)

RBC: Red blood cells count (*106/μl)                                                                         WBC: Total leucocyte count (*103/μl)

Mono: Monocytes (%)                                                                                         PCV: Packed cell volume (%)

MCHC: Mean cell haemoglobin conc. (g%)                                                                     a, b, c:   significantly different from control, p0.05, p0.01, p0.001

Table 2. Group mean bodyweights for rats dosed with HHCB
Dose group Body weight (g) at week
Males 0 4 8 13 0-13 week gain
Control 218 ± 19 389 ± 42 481 ± 53 544 ± 65 326 ± 52
5 224 ± 15 426 ± 29 533 ± 41 611 ± 55 388 ± 47
15 225 ± 13 411 ± 22 505 ± 28 573 ± 35 348 ± 30
50 224 ± 15 405 ± 33 499 ± 48 559 ± 54 335 ± 46
150 221 ± 17 403 ± 35 495 ± 44 561 ± 52 340 ± 45
Females
Control 177 ± 13 262 ± 30 299 ± 35 323 ± 36 146 ± 26
5 175 ± 09 254 ± 26 290 ± 31 314 ± 33 139 ± 30
15 170 ± 13 245 ± 18 285 ± 22 309 ± 26 139 ± 26
50 175 ± 14 248 ± 25 287 ± 30 314 ± 30 139 ± 24
150 173 ± 11 250 ± 26 280 ± 25 305 ± 26 132 ± 17

table 3. Significant blood chemistry parameters for rats treated with HHCB at weeks 7 and 13
Dose group (mg/kg) Glucose mg% Triglycerides mg%
Week 7 Week 13 Week 7 Week 13
Males
Control 116±12,1 117±7,3 84±27,2 95±32
5 121±10,1 130±8,9 83±23,7 105±35,1
15 105±13.0** 113±8 68±16.7* 66±20.1**
50 105±7.9** 111±9.8* 62±20.5** 58±24.8***
150 104±6.9** 109±8.0* 58±11.4** 55±7.1***
Females
Control 116±13,2 113±10 55922,7 58±22,1
5 117±8,4 113±9,1 57±34,5 56±26,5
15 107±12.4* 115±9,1 43±9,1 53±11,6
50 108±13.4* 109±17,3 55±21,3 56±13,8
150 104±9.8** 107±9,2 48±19,9 53±24,8
* Significantly different from control, P <0,05
** Significantly different from control, P <0,01
*** Significantly different from control, P <0,001
Table 4. Group mean absolute liver weight, body weight related liver weights and brain related liver weights for rats sacrificed after 13 weeks of treatment with HHCB.
Absolute liver weight (g) Liver weights relative to body weight (5) Liver weights relative to brain weight (%)
Males
Control 15,7 ± 2,5 3,1 ± 0,5 741,8 ± 120,2
5 20,2 ± 4,1 3,6 ± 0,9 957 ± 201.8*
15 17,4 ± 2,3 3,3 ± 0,4 829,8 ± 102.8*
50 18,8 ± 2,3 3,6 ± 0,3 875,8 ± 103.5*
150 19,3 ± 1,6 3,7 ± 0,2 913,7 ± 82.1*
Females
Control 9,8 ± 01 3,3 ± 0,3 492,1 ± 51,4
5 9,9 ± 0,7 3,4 ± 0,3 504,8 ± 42
15 9,8 ± 1,1 3,4 ± 0,3 486,7 ± 46,1
50 9,8 ± 1,2 3,5 ± 0,5 497,7 ± 64,2
150 10,3 ± 1,2 3,7 ± 0,3 519,5 ± 53,4
* significantly different from control, P0,01

Conclusions:
It is concluded there were no significant adverse effects at any dose level up to the highest tested level of 150 mg/kg bw/day
Executive summary:

A 13-week oral toxicity study in accordance with OECD guideline 408 and conforming to GLP was conducted in 150 Crl:CD (SD)Br rats (5 groups of 15 males (weight 182-260 g) and 15 females (weight 152 and 201 g)). They received HHCB (purity not reported in report, confirmed to be >95% pure undiluted material, IFF personal communication with RIFM) by dietary admixture at 0, 5, 15, 50, or 150 mg/kg bw/day. HHCB was added to the diet. Analyses of diet indicated that desired homogeneity was reached. The concentrations of HHCB in the test diets were adjusted weekly based on bodyweight and food consumption from the previous week. The mean achieved daily intakes were 5.4, 15.7, 51.8 and 155.8 mg HHCB/kg bw for males and 5.1, 15.6, 51.9 and 154.6 mg HHCB/kg bw for females. After the treatment period, 3 males and 3 females from the control and the high dose groups were maintained for a treatment-free period of 4 weeks.

Observations included mortality and clinical signs (daily), body weight and food consumption (weekly), ophthalmoscopy (before start, at week 13 and at the end of the treatment-free period, only controls and high dose animals), urinalysis (at week 6 and 12 of treatment and at the end of the treatment-free period), haematology and clinical chemistry (at week 7 and 13 of treatment and at the end of the recovery period), macroscopy, organ weights and histopathology (on all tissues from controls and high dose animals, on all gross lesions, and on lungs, liver, kidneys and male and female reproductive and accessory organs from all animals).

There were no mortalities or adverse clinical signs. Body weight and food consumption of treated groups were similar to those observed in the control group. No changes in ophthalmologic evaluation were observed and no significant histopathological findings at any dose in any tissue including  reproductive organs and bone marrow. (Api and Ford, 1999; Hopkins et al., 1996).

A variety of statistically significant differences between control and test animals were seen in haematology and blood chemistry although these differences were all small, often not proportional to dose, often seen only at one time point and/or in one sex, and, with two exceptions, well within historical controls. There were no significant differences seen at the end of the treatment-free period. A statistically significant higher mean partial thromboplastin time (PTT) was seen in the females at 150 mg/kg bw/day (16.6 sec.) and 50 mg/kg bw/day (16.5 sec.) compared to controls (14.3 sec) at week 13. These values were slightly higher than the high end of the historical controls. The increase in PTT can indicate acquired hypofibrinogenemia. However, in this study, the abnormal height of control fibrinogen might explain the increase in PTT. Moreover, there is no clear dose-response relationship, a lack of other corresponding findings such as effects on liver cells (absence of histomorphological alterations, absence of liver enzyme activation), a lack of any other indication of abnormal (retarded) coagulation and no signs of uncontrolled spontaneous bleeding such as abnormal occurrence of petechia or hemorrhagia in organs, skin or mucous membranes. There were no significant differences in prothrombin times in any of the groups (males and females) in comparison with their respective controls. All groups of treated females had statistically significant lower fibrinogen than controls, without a clear doseresponse, while the mean fibrinogen level for the female control group was significantly higher than the highest historical control value. At the end of the treatment-free period, no significant effect was found on either fibrinogen, partial thromboplastin time or prothrombin time in females nor males. For these reasons, the deviations in PTT are not considered of toxicological relevance.

The mean red blood cell counts in males but not females at week 7 in the 150 and 50 mg/kg bw/day groups were significantly slightly lower than in the controls. At week 13, the males did not differ significantly from the controls in any group however the high dose females were slightly lower than controls.

The total leucocyte count was significantly lower for the females in the 15, 50 and 150 mg/kg bw/day groups compared to controls at week 7, without any dose-response relationship.

Statistically significant lower monocytes were reported for males, but not females, at all but the low dose group at week 13 but not 7. The imprecision of the reported values makes significance questionable.

The packed cell volumes for males at 50 and 150 mg/kg bw/day were statistically significantly lower than controls at week 7 but not week 13. There was no correlation with dose. The mean haemoglobin concentrations for all dosed groups at week 13, all dosed females at week 7 and for the two highest dosed male groups at week 7 were statistically significantly higher than the respective controls but with no correlation with dose and all values, except for that of the males at 50 mg/kg bw/day at week 7, were within the historical control ranges.

ALT levels were statistically significantly lower in females but not males in the two higher dosed groups at week 7. They were not significantly different in males or females at week 13. AST levels were statistically significantly lower than controls in males at all dose levels but only in the highest dosed females at week 7. However, at week 13, all but the lowest dosed females had statistically significantly lower AST levels with no significant differences in any male group.

There were statistically significant lower triglyceride levels relative to controls seen in the males at all but the lowest dose group (5 mg/kg bw/day) at both week 7 and week 13 and the values were dose related. There were no significant differences with females. There were, however, statistically significant lower glucose levels in all but the lowest dose group in both males and females at week 7 but with no clear dose relationship. At week 13, the females and males at 50 and 150 mg/kg bw/d were still lower but statistically significantly only in the males. While these differences appear to be attributable to HHCB administration, they are considered of minor toxicological significance and likely reflect slight nutritional effects.

There were no statistically significant differences in any haematological parameter or blood chemistry values between test and control animals at the end of the treatment free period (Api and Ford, 1999b; Hopkins et al., 1996).

The haematology and blood chemistry effects were all small, often not proportional to dose, often seen only at one time point and/or in one sex, and, with two exceptions, well within historical controls and are not considered to “reduce the capacity of an organism or acomponent of an organism to function in a normal manner” (Abadin et al., 1998). This, and the fact that these findings were not accompanied by any adverse histopathology (e.g. in the bone marrow) or other related findings, leads to the conclusion that they are not adverse effects.

It is concluded there were no significant adverse effects at any dose level up to the highest tested level of 150 mg/kg bw/day (NOAEL ≥ 150 mg/kg bw/day).

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
150 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
The quality of the database is high because the information fulfils the REACH requirements: a 90-day study according to OECD guideline (and GLP)

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed

Additional information

Key study: A 13-week oral toxicity study in accordance with OECD guideline 408 and conforming to GLP was conducted in 150 Crl:CD (SD)Br rats (5 groups of 15 males (weight 182-260 g) and 15 females (weight 152 and 201 g)). They received HHCB (purity not reported in report, confirmed to be >95% pure undiluted material, IFF personal communication with RIFM) by dietary admixture at 0, 5, 15, 50, or 150 mg/kg bw/day. HHCB was added to the diet. Analyses of diet indicated that desired homogeneity was reached. The concentrations of HHCB in the test diets were adjusted weekly based on bodyweight and food consumption from the previous week. The mean achieved daily intakes were 5.4, 15.7, 51.8 and 155.8 mg HHCB/kg bw for males and 5.1, 15.6, 51.9 and 154.6 mg HHCB/kg bw for females. After the treatment period, 3 males and 3 females from the control and the high dose groups were maintained for a treatment-free period of 4 weeks.

Observations included mortality and clinical signs (daily), body weight and food consumption (weekly), ophthalmoscopy (before start, at week 13 and at the end of the treatment-free period, only controls and high dose animals), urinalysis (at week 6 and 12 of treatment and at the end of the treatment-free period), haematology and clinical chemistry (at week 7 and 13 of treatment and at the end of the recovery period), macroscopy, organ weights and histopathology (on all tissues from controls and high dose animals, on all gross lesions, and on lungs, liver, kidneys and male and female reproductive and accessory organs from all animals).

There were no mortalities or adverse clinical signs. Body weight and food consumption of treated groups were similar to those observed in the control group. No changes in ophthalmologic evaluation were observed and no significant histopathological findings at any dose in any tissue including  reproductive organs and bone marrow. (Api and Ford, 1999; Hopkins et al., 1996).

A variety of statistically significant differences between control and test animals were seen in haematology and blood chemistry although these differences were all small, often not proportional to dose, often seen only at one time point and/or in one sex, and, with two exceptions, well within historical controls. There were no significant differences seen at the end of the treatment-free period. A statistically significant higher mean partial thromboplastin time (PTT) was seen in the females at 150 mg/kg bw/day (16.6 sec.) and 50 mg/kg bw/day (16.5 sec.) compared to controls (14.3 sec) at week 13. These values were slightly higher than the high end of the historical controls. The increase in PTT can indicate acquired hypofibrinogenemia. However, in this study, the abnormal height of control fibrinogen might explain the increase in PTT. Moreover, there is no clear dose-response relationship, a lack of other corresponding findings such as effects on liver cells (absence of histomorphological alterations, absence of liver enzyme activation), a lack of any other indication of abnormal (retarded) coagulation and no signs of uncontrolled spontaneous bleeding such as abnormal occurrence of petechia or hemorrhagia in organs, skin or mucous membranes. There were no significant differences in prothrombin times in any of the groups (males and females) in comparison with their respective controls. All groups of treated females had statistically significant lower fibrinogen than controls, without a clear doseresponse, while the mean fibrinogen level for the female control group was significantly higher than the highest historical control value. At the end of the treatment-free period, no significant effect was found on either fibrinogen, partial thromboplastin time or prothrombin time in females nor males. For these reasons, the deviations in PTT are not considered of toxicological relevance.

The mean red blood cell counts in males but not females at week 7 in the 150 and 50 mg/kg bw/day groups were significantly slightly lower than in the controls. At week 13, the males did not differ significantly from the controls in any group however the high dose females were slightly lower than controls.

The total leucocyte count was significantly lower for the females in the 15, 50 and 150 mg/kg bw/day groups compared to controls at week 7, without any dose-response relationship.

Statistically significant lower monocytes were reported for males, but not females, at all but the low dose group at week 13 but not 7. The imprecision of the reported values makes significance questionable.

The packed cell volumes for males at 50 and 150 mg/kg bw/day were statistically significantly lower than controls at week 7 but not week 13. There was no correlation with dose. The mean haemoglobin concentrations for all dosed groups at week 13, all dosed females at week 7 and for the two highest dosed male groups at week 7 were statistically significantly higher than the respective controls but with no correlation with dose and all values, except for that of the males at 50 mg/kg bw/day at week 7, were within the historical control ranges.

ALT levels were statistically significantly lower in females but not males in the two higher dosed groups at week 7. They were not significantly different in males or females at week 13. AST levels were statistically significantly lower than controls in males at all dose levels but only in the highest dosed females at week 7. However, at week 13, all but the lowest dosed females had statistically significantly lower AST levels with no significant differences in any male group.

There were statistically significant lower triglyceride levels relative to controls seen in the males at all but the lowest dose group (5 mg/kg bw/day) at both week 7 and week 13 and the values were dose related. There were no significant differences with females. There were, however, statistically significant lower glucose levels in all but the lowest dose group in both males and females at week 7 but with no clear dose relationship. At week 13, the females and males at 50 and 150 mg/kg bw/d were still lower but statistically significantly only in the males. While these differences appear to be attributable to HHCB administration, they are considered of minor toxicological significance and likely reflect slight nutritional effects.

There were no statistically significant differences in any haematological parameter or blood chemistry values between test and control animals at the end of the treatment free period (Api and Ford, 1999b; Hopkins et al., 1996).

The haematology and blood chemistry effects were all small, often not proportional to dose, often seen only at one time point and/or in one sex, and, with two exceptions, well within historical controls and are not considered to “reduce the capacity of an organism or a component of an organism to function in a normal manner” (Abadin et al., 1998). This, and the fact that these findings were not accompanied by any adverse histopathology (e.g. in the bone marrow) or other related findings, leads to the conclusion that they are not adverse effects.

It is concluded there were no significant adverse effects at any dose level up to the highest tested level of 150 mg/kg bw/day (NOAEL ≥ 150 mg/kg bw/day). (Hopkins et al., 1996, Api and Ford, 1999)

 

Additional studies: Three dermal subchronic studies are available. In two of these there was some evidence of liver weight increases (at 100 mg/kg bw/day for 13 weeks) and body weight decreases (at 36 mg/kg bw/day for 26 weeks) but the magnitude of these effects were not reported and their significance cannot be determined. (Gressel et al., 1980(1), Gressel et al., 1980(2)) In a third dermal 26-week study, no effects were seen up to and including the highest dose administered (200 mg/kg bw/day). However, because 1) neither collars nor occlusion were used to prevent oral intake making it impossible to determine actual exposures, 2) the area of application was not reported, and 3) the lack of significance in the findings reported in the first two studies and the lack of an adverse effect dose in the third, it is impossible to conclude a true NOAEL in terms of dermal toxicity. (Estes et al., 1980)

When administered as part of a fragrance mixture, inhalation exposure to HHCB up to a maximum tested dose of 132 μg/m3 for 4 hr per day, 5 days per week for 13 weeks did not result in any toxicity. This study is of limited value because HHCB was only present at rather low levels in the mixtures. (Fukayama et al., 1999)

 

Conclusion and discussion: In an adequate 90-day oral study, there were no mortalities or adverse clinical signs. Body weight and food consumption of treated groups were similar to those observed in the control group. No changes in ophthalmologic evaluation were observed and no significant histopathological findings at any dose.

The haematology and blood chemistry differences from controls were all small, often not proportional to dose, often seen only at one time point and/or in one sex, and, with two exceptions, well within historical controls and are not considered to “reduce the capacity of an organism or a component of an organism to function in a normal manner” (Abadin et al., 1998). This, and the fact that these findings were not accompanied by any adverse histopathology or other related findings, leads to the conclusion that they are not adverse effects. A NOAEL of ≥150 mg/kg bw/day, the highest dose tested, for HHCB in rats is concluded.

 

(Summary in line with EU-RAR, 2008)


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
One 90-d oral toxicity study available, supported by the dose range finding study. The study was conducted in accordance with OECD guideline 408 and under GLP conditions

Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:
Supporting study of limited value because HHCB was tested as part of a mixture and only present at rather low levels.

Justification for selection of repeated dose toxicity inhalation - local effects endpoint:
Supporting study of limited value because HHCB was tested as part of a mixture and only present at rather low levels.

Justification for selection of repeated dose toxicity dermal - systemic effects endpoint:
Three supporting studies are available in which no effects were observed, however due to shortcomings in the test method, they are of limited value.

Justification for selection of repeated dose toxicity dermal - local effects endpoint:
Three supporting studies are available in which no effects were observed, however due to shortcomings in the test method, they are of limited value.

Justification for classification or non-classification

Based on the available repeated dose toxicity studies, there is no need to classify HHCB, in accordance with 67/548/EEC and 1272/2008/EC.