Mono- and/or di- and/or tri(1-phenylethyl)-m-cresol and p-cresol

Endpoint summary

Description of key information

In a 28-day repeated dose toxicity study of Atlen SK on rats NOAEL of 1000 mg/kg bw/day was identified with respect to repeated dose toxicity effects.

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:

Jan-Aug 2012

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

yes

Remarks:

All the deviations that happened were reported to Study director, and records of the deviations are enclosed in the study raw data file. The integrity of the study was not affected.

Principles of method if other than guideline:

Deviations recorded during the study:
- Different body weight values of females at the start of the study (February 2, 2012): the females prepared for the study had higher body weight than that planned in the study plan; during the randomization, the animals were divided into groups for a balanced body weight mean in all groups
- The measurements for clinical chemistry not performed in some animals (June 12, 2012): in the cases of insufficient serum sample volume for clinical chemistry evaluation, sufficient information for statistical evaluation was obtained from other animals
- Temporary oscillations of the temperature and humidity in the experimental room (July 11, 2012): minor deviations in the relative air humidity and temperature were recorded during the study, but they have not influenced the health condition of the animals
- Sending of samples for re-analysis (August 15, 2012): during the study, samples were not sent to the Sponsor for re-analysis in accordance with the study plan.

GLP compliance:

yes (incl. QA statement)

Species:

rat

Strain:

Wistar

Sex:

male/female

Route of administration:

other: Each animal from the dose groups received orally the appropriate dose of the Test item as suspension in the volume of 1 mL/100 g b.w. . Individual doses were adjusted according to the weekly body weight.

Vehicle:

other: 95% Methylcellulose 0.5 % +5% sunflower oil

Duration of treatment / exposure:

90 days

Frequency of treatment:

Dosing: Daily for 90 consecutive days

Remarks:

Doses / Concentrations:
The concentrations of the Test item in the emulsions for dose groups D1, D2 and D3 were 0.5 %, 12.5 % and 30 %, respectively (50, 125, 300 mg/kg).
Basis:
nominal in diet

No. of animals per sex per dose:

group C, D3: 20 males + 20 females
group D1, D2: 10 males + 10 females

Control animals:

other: Yes: The control animals were treated with the vehicle in the same regime and volume.

Dose descriptor:

NOAEL

Effect level:

> 300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Critical effects observed:

not specified

No clinical signs of intoxication and changes in health condition were observed during acclimation and administration periods in control group C and treated groups D1 (50 mg/kg of body weight), D2 (125 mg/kg of body weight) and D3 (300 mg/kg of body weight).

 

The individual values of body weight increased normally according to the animals’ age.

 

The oscillation of food consumption in all four groups during the study did not affect the growth of animals and the increase of body weight in all the animals. 

 

A decrease in the mean corpuscular volume values of erythrocytes was recorded in Examination 2, but this decrease influenced neither the changes in red blood cell count nor those in the amount of hemoglobin. Thus this finding was biologically insignificant. No treatment related changes were documented in white blood cell parameters, coagulation parameter of bone marrow count after 90-day repeated administration of Test item.

 

No treatment related changes of clinical chemistry parameters were observed after 90-day oral administration of Atlen SK. The relation of the increase in the triglycerides in the recovery D3 animals to Atlen SK administration is not probable.

 

No treatment related changes were found in urine parameters monitored after 90-day oral administration of Atlen SK.

 

None of the animals in this study showed any ophthalmologic findings, both before and at the end of the observation period (before necropsy).

 

Organ weight analysis did not find any treatment-related changes.

 

Athlen SK in doses used did not cause gross or histopathological changes in the liver and kidneys of the animals indicative of a toxic effect.

Atlen SK orally administered to rats did not cause any pathological changes in the gastrointestinal tract.

Other lesions found in the treated animals were either of spontaneous character or they were not in direct relation with the Atlen SK administered.

Conclusions:

Based on available data Atlen SK is not classified as toxic after repeated exposure via oral route (STOT RE 2 according to CLP or R48/22 according to DSD).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

300 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Aug 2008 - Mar 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: this study was planned and executed in accordance with relevant guidelines as well as the requirements of the GLP

Qualifier:

according to guideline

Guideline:

EU Method B.9 (Repeated Dose (28 Days) Toxicity (Dermal))

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

yes

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: conventional husbandry of Institute of Occupational Medicine, Łódź, Poland
- Age at study initiation: 9 weeks
- Weight at study initiation: see Tables 2 and 3 in section Any other information on results incl. tables
- Fasting period before study: not reported
- Housing: The animals were kept in plastic cages with wired lid (dimensions: 58 x 37 x 21 cm), 5 animals per cage, each sex separately. UV-sterilised wooden shavings were used as bedding and were changed twice a week.
- Diet (e.g. ad libitum): ad libitum, The animals were given standard granulated “Murigran” laboratory fodder produced by Wytwórnia Koncentratów i Mieszanek Paszowych AGROPOL, Motycz, Poland
- Water (e.g. ad libitum): ad libitum. The animals were given tap water
- Acclimation period: prior to the experiment the animals were quarantined for at least 5 days

The animals were divided into 2 experimental groups: one control group (group 0) and one group treated with the test item (group 1). Two concurrent satellite groups were conducted: treated (group 1 SAT) and control (group 0 SAT).

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 - 23
- Humidity (%): 45 - 65
- Air changes (per hr): not reported
- Photoperiod (hrs dark / hrs light): 12 / 12

Type of coverage:

other: gauze patch

Vehicle:

unchanged (no vehicle)

Details on exposure:

TEST SITE
- Area of exposure: not reported
- % coverage: ca. 10
- Type of wrap if used: elastic band and plaster
- Time intervals for shavings or clipplings: shavings were performed 24 hrs before the start of the experiment and then in a week-long periods

REMOVAL OF TEST SUBSTANCE
- Washing (if done): residues of the test item were removed with the aid of a tampon with oil
- Time after start of exposure: each day after termination of the 6 hours daily exposure period

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 1000 mg/bw
- Constant volume or concentration used: in order to keep constant level of dosage the amounts of the test item were adjusted to body weight changes on days of weighing

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

6 hrs per day for 28 days

Frequency of treatment:

once a day for 6 hours

Remarks:

Doses / Concentrations:
1000 mg/kg bw
Basis:
nominal per unit body weight

No. of animals per sex per dose:

6 animals per sex per dose

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale: dose was selected on the basis of results of an earlier acute dermal toxicity study during which no effects were seen at the limit dosing of 2000 mg/kg bw
- Rationale for selecting satellite groups: satellite groups were chosen in accordance with the requirements of the guideline
- Post-exposure recovery period in satellite groups: 14 days

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily (all animals were observed for mortality/morbidity twice a day during week and once a day during days off)
- Cage side observations checked in Table 2 in the section Any other information on materials and methods were included.
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once a day

DERMAL IRRITATION (if dermal study): Yes
- Time schedule for examinations: once a day

BODY WEIGHT: Yes
- Time schedule for examinations: bw was controlled prior to the beginning of the experiment and once a week thereafter

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of the experiement
- Anaesthetic used for blood collection: Yes (xylazine-ketamine mixture at dose 10 mg/kg bw of xylazine and 100 mg/kg bw of ketamine)
- Animals fasted: Yes (about 18 hrs long period of fasting was employed)
- How many animals: all animals (48)
- Parameters checked in Table 1 in the section Any other information of materials and methods were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of the experiement
- Animals fasted: Yes (about 18 hrs long period of fasting was employed)
- How many animals: all animals (48)
- Parameters checked in Table 1 in the section Any other information of materials and methods were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

OTHER: None

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see Table 3 in the section Any other information of materials and methods)
HISTOPATHOLOGY: Yes (see Table 3 in the section Any other information of materials and methods )

Statistics:

Results were elaborated using one-way variance analysis and t-Student test (body weight of animals, food consumption) as well as Dunnet test (results of clinical-chemical investigations, internal organ weights) with p =< 0.05.

Clinical signs:

no effects observed

Description (incidence and severity):

for details see section Details on results

Dermal irritation:

no effects observed

Description (incidence and severity):

for details see section Details on results

Mortality:

no mortality observed

Description (incidence):

for details see section Details on results

Body weight and weight changes:

no effects observed

Description (incidence and severity):

for details see section Details on results

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

for details see section Details on results

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

for details see section Details on results

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

for details see section Details on results

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

for details see section Details on results

Gross pathological findings:

no effects observed

Description (incidence and severity):

for details see section Details on results

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

for details see section Details on results

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

for details see section Details on results

Details on results:

Note: Tables can be found in the sections Any other information of results incl. tables (Tables 1-29) and Overall remarks (Tables 30-47).

CLINICAL SIGNS AND MORTALITY
The clinical signs stated in animals are presented in detail in Table 1.
The observed signs were transient. 3-day desquamation of epidermis was observed in two females (No 13 and No 14) of group 1 during second week of experiment. 3-day respiratory murmurs were stated in one female (No 14) of control group during the first week of the experiment. Respiratory murmurs were observed in three males of group 1 SAT: in male No 9 – lasting 2 days during the 1st week of the experiment; in male No 8 – lasting 2 days during the 3rd week of the experiment; in male No 11 – lasting 1 day during the 3rd week of the experiment.

No deaths of animals were stated during experiment.

BODY WEIGHT AND WEIGHT GAIN
Transient body weight loss during experiment was stated in some animals: in two females of group 0 (No 14 and No 18), one female of group 0 SAT (No 21), two males of group 1 (No 1 and No 4), one female of group 1 (No 14), two males of group 1 SAT (No 7 and No 8) and one female of group 1 SAT (No 22). Constant body weight loss from 2nd week to the end of the experiment was stated in two males of group 1 (No 2 and No 5) (Table 1).
Average body weights of animals determined in week-long periods are presented in Table 2 (males) and Table 3 (females). Average body weight of rat males and females of treated group did not differ statistically significantly from the average body weight of animals of control group. Average body weights of animals of satellite groups are presented in Table 4 (males) and Table 5 (females).
During 28-day period of test item application and during 14-day period of additional observation average body weight of males and females of treated group was level with the average body weight of males and females of control group.

FOOD CONSUMPTION
The average food consumption of animals of treated and control group is presented in Table 6 (males) and Table 7 (females).
The average food consumption of males and females of treated group was level with the control group.
The average food consumption of animals of satellite groups is presented in Table 8 (males) and in Table 9 (females).
During the first week of the experiment statistically significant lower food consumption was stated in males of group 1 SAT compared with control group. During the other periods in case of treated males and during the entire experiment in case of treated females the food consumption was comparable with food consumption in control group.
After termination of test item administration, during 14-day period of additional observation, the average food consumption of males and females of group 1 SAT was level with the average food consumption of males and females of group 0 SAT.

FOOD EFFICIENCY - not examined
WATER CONSUMPTION - not examined
OPHTHALMOSCOPIC EXAMINATION - not examined

HAEMATOLOGY
-Circulatory blood investigation
Results of circulatory blood investigation are presented in Tables 10-17.
Average values of results of circulatory blood investigation of males and females treated with Atlen SK and of control groups were similar, without statistically significant differences (Table 10, 11).
-Coagulological investigation
Prothrombin time was determined during coagulological investigation of all animals. No statistically significant differences were revealed in case of this investigation (Table 10, 11, 12, 13).
-Bone marrow examination
Results of bone marrow examination are presented in Tables 18-29.
In erythrocyte system average number of particular cells in animals of treated group did not differ statistically significantly from the averages of control group. The exception was decrease in number of polychromatic erythroblasts in males (Table 18). No statistically significant differences were stated in erythrocyte system in satellite group (Table 20, 21).
In leukocyte system of males of group 1 increase in number of neutrophilic segmented granulocytes was stated (Table 22). No statistically significant changes were stated in females of group 1 (Table 23). No statistically significant changes in leukocyte system were stated in males
and females of satellite group (Table 24, 25).
No statistically significant changes in content of different cells were stated in treated group and treated satellite group (Tables 26-29).

CLINICAL CHEMISTRY
-Biochemical investigation
Results of biochemical investigation are presented in Tables 30-33.
No statistically significant differences were stated in the scope of biochemical investigations in treated group of males and females compared with the control (Table 30, 31). Statistically significant changes did not occur in biochemical investigations of satellite group (Table 32, 33).
-Enzymatic investigation
Results of enzymatic investigation are presented in Tables 34-37.
Activity of investigated enzymes in males and females of treated group did not differ statistically significantly from results obtained in control group (Table 34, 35). No statistically significant changes in activity of enzymes were observed in group 1 SAT, except for decrease in AP activity in females (Table 36, 37).

URINALYSIS - not examined
NEUROBEHAVIOUR - not examined

ORGAN WEIGHTS
Average absolute weight of internal organs of animals is presented in Tables 39-42.
Statistically significant changes in absolute weight of internal organs in form of increase in weight of liver, kidneys and adrenals were stated in females of group 1. No statistically significant changes in internal organs’ weight were stated in satellite groups.
Average relative weight of internal organs of animals is presented in Tables 43-46.
In scope of relative values increase in weight of adrenals occurred in males and females of group 1, increase in weight of liver and kidneys occurred in females of group 1 and increase in weight of ovaries occurred in females of group 1 SAT.

GROSS PATHOLOGY
List of gross changes stated in animals is presented in Table 38.
Enlargement of submandibular lymph nodes was stated in one male (No 4) of group 1.

HISTOPATHOLOGY: NON-NEOPLASTIC
List of histopathological changes stated in animals is presented in Table 47.
During histopathological examination the changes were stated in the following organs:
- in lungs: emphysema in two males (No 2 and No 4) of group 1 and in one male (No 7) of group 1 SAT; erythrocytorrhagia in two males (No 1 and No 5) and two females (No 15 and No 16) of group 0 as well as in two males (No 4 and No 5) and two females (No 13 and No 15) of group 1.
- in kidneys: dilatation of renal pelvis and slight atrophy of pulp in kidney in male (No 3) of group 0 and female (No 18) of group 1.
- in lymph nodes: overgrowth and purulent inflammation of submandibular lymph node in male (No 4) of group1.

OTHER FINDINGS - none were reported

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Critical effects observed:

not specified

Results interpreatation

No significant differences in appearance and behaviour of animals of treated and control groups were stated in the study. Respiratory murmurs observed in some animals were transient and occurred also in control group. This sign could be caused by infection of upper respiratory tract which occurs quite often in laboratory animals. Desquamation of epidermis observed in two females was caused by topical skin irritation by test item. This sign was transient and was observed only for 3 days during the second week of the experiment. Transient body weight losses occurred in nine animals of different groups. Lower food consumption which was stated in males of group 1 SAT during first week of experiment did not cause statistically significant body weight loss in these males. Decrease in food consumption in males of group 1 SAT should be considered as accidental one since there was no statistically significant lower food consumption in group 1 during the first week of the experiment.

None of animals died during the entire experiment.

No statistically significant differences were recorded in haematological investigation of circulatory blood.

Two statistically significant changes occurred in bone marrow examination.

The first one concerned decrease in number of polychromatic erythroblasts in males of treated group. One may consider this change as an accidental and not connected with the test item because number of orthochromatic erythroblasts which arise from polychromatic erythroblasts and number of red cells in circulatory blood did not differ statistically significantly from the control group. The second change concerning increase in number of neutrophilic segmented granulocytes in males of group 1 is not confirmed in the following stages of leukocyte system development and should be considered as an accidental one.

On the ground of statistical analysis of biochemical investigations no statistically significant differences were stated; whereas in enzymatic investigation decrease in AP activity occurred in females of group 1 SAT. Taking into account that activity of this enzyme is almost identical in females of both group 1 SAT and group 1 this change is probably not connected with the test item. It was caused by higher AP activity in females of group 0 SAT in comparison to females of group 0. It is worth to mention that in some samples hemolysis occurred and it could influence results of AP activity and decreased diagnostic value of obtained results.

In spite of occurrence of single statistically significant changes which were probably independent of test item, on the ground of statistical analysis of clinical-chemical investigation, one may accept that Atlen SK does not harmfully influence the animals.

Some pathological changes in lungs, kidneys and lymph nodes occurred in single animals of group 0, group 1 and group 1 SAT. Results of histopathological examination of changes in organs did not reveal any causal connection with the test item. Some changes i.e. purulent adenitis could be caused by microbial infection. Changes concerning lungs (erythrocytorrhagia, emphysema) could be caused by reaction to anaesthetic which causes often depression of respiratory-circulatory system, sudden cramp of bronchi and in consequence – erythrocytorrhagia and emphysema of lungs.

In histopathological examination no differences were stated in treated and untreated skin.

The statistically significant differences in absolute and relative weight of some organs are not confirmed histopathologically and should not be connected with the test item influence.

The post mortem examination indicates that Atlen SK given dermally to rats in dose of 1000 mg/kg b.w. for 28 days does not cause any specific pathological changes in organs and tissues of animals.

Table 1

Atlen SK.28-day repeated dose dermal toxicity study on rats.

Clinical signs in animals - overall list

	0		0 SAT		1		1 SAT
	males	females	males	females	males	females	males	females
Test animals	6	6	6	6	6	6	6	6
Animals with clinical signs	-	1 [14]*	-	-	-	2 [13,14]*	3 [8,9,11]*	-
SKIN AND HAIR
Desquamation of epidermis	-	-	-	-	-	2 [13,14]*	-	-
RESPIRATORY SYSTEM
Respiratory murmurs	-	1 [14]*	-	-	-	-	3 [8,9,11]*	-
Body weight loss	-	2 [14,18]*	-	1 [21]*	4 [1,4]* [2,5]	1 [14]*	2 [7,8]*	1 [22]*

[ ] – computer numbers of animals in which clinical signs were observed

* – clinical signs which were observed transiently

 

Table 2 and Table 3

Atlen SK.28-day repeated dose dermal toxicity study on rats.

Average body weight [g] – males and females

Week	males of GROUP		females of GROUP
	0	1	0	1
0	240,3 ± 11,79	240,3 ± 12,91	170,7 ± 7,15	170,7 ± 7,74
1	258,7 ± 9,52	241,5 ± 34,05	165,8 ± 14,43	170,2 ± 10,23
2	269,3 ± 8,12	260,3 ± 38,86	180,5 ± 6,95	182,3 ± 8,55
3	287,2 ± 11,69	278,3 ± 46,34	188,2 ± 11,65	187,8 ± 11,87
4	288,3 ± 14,79	266,7 ± 49,84	189,8 ± 8,42	189,0 ± 7,13

 

Table 4 and Table 5

Atlen SK.28-day repeated dose dermal toxicity study on rats.

Average body weight [g] – males and females (satellite groups)

Week	males of GROUP		females of GROUP
	0 SAT	1 SAT	0 SAT	1 SAT
0	240,2 ± 10,09	240,2 ± 14,96	170,7 ± 7,58	170,7 ± 9,35
1	260,5 ± 9,52	241,0 ± 17,05	172,5 ± 12,32	173,2 ± 12,92
2	277,3 ± 10,33	265,7 ± 18,04	189,2 ± 15,87	182,5 ± 19,40
3	294,3 ± 15,23	288,5 ± 15,66	192,5 ± 17,44	190,8 ± 16,19
4	304,8 ± 19,84	298,8 ± 17,86	203,7 ± 18,32	199,2 ± 11,44
5	317,7 ± 19,93	315,5 ± 16,93	214,3 ± 20,29	209,8 ± 11,34
6	320,7 ± 17,81	318,3 ± 19,69	216,5 ± 25,84	212,2 ± 15,38

 

Table 6 and Table 7

Atlen SK.28-day repeated dose dermal toxicity study on rats.

Average food consumption [g/rat/day] – males and females

Week	males of GROUP		females of GROUP
	0	1	0	1
1	23,8 ± 2,16	21,0 ± 6,09	14,2 ± 2,18	16,6 ± 1,01
2	22,1 ± 1,27	21,9 ± 5,30	15,9 ± 0,87	16,5 ± 1,14
3	22,3 ± 1,62	23,6 ± 3,29	16,6 ± 2,28	16,1 ± 0,98
6	23,6 ± 1,63	21,6 ± 5,21	17,8 ± 2,61	17,6 ± 0,78

 

Table 8 and Table 9

Atlen SK.28-day repeated dose dermal toxicity study on rats.

Average food consumption [g/rat/day] - males and females (satellite groups)

Week	males of GROUP		females of GROUP
	0 SAT	1 SAT	0 SAT	1 SAT
1	3,9 ± 1,14	20,3 ± 1,84*	16,5 ± 1,06	16,6 ± 1,86
2	23,3 ± 1,37	21,9 ± 1,74	16,7 ± 1,43	16,8 ± 2,06
3	23,2 ± 1,16	23,6 ± 2,21	16,9 ± 1,75	17,0 ± 1,13
4	23,2 ± 1,59	25,0 ± 4,66	17,8 ± 2,12	18,6 ± 1,49
5	24,2 ± 1,74	24,3 ± 1,57	18,8 ± 1,94	19,4 ± 1,08
6	25,2 ± 1,62	24,9 ± 1,01	19,4 ± 3,88	19,6 ± 1,33

* – statistically significant difference with p =< 0.05

 

Table 10 and Table 11

Atlen SK.28-day repeated dose dermal toxicity study on rats.

Results of haematological investigation – males and females

Study type	males of GROUP		females of GROUP
	0	1	0	1
HAEMOGLOBIN g/l HAEMATOCRIT 1/1 ERYTHROCYTES x 10^12/l MCV fl MCH pg MCHC g/l THROMBOCYTES x 10^9/l LEUKOCYTES x 10^9/l PROTHROMBIN TIME s	146,17 ± 17,98 0,45 ± 0,05 8,19 ± 0,85 54,83 ± 1,47 17,82 ± 0,58 326,00 ± 2,97 486,67 ± 222,29 7,28 ± 2,43 13,63 ± 0,77	144,50 ± 13,52 0,44 ± 0,05 7,90 ± 0,90 55,83 ± 2,14 18,33 ± 0,70 327,83 ± 7,28 580,00 ± 270,28 7,43 ± 2,71 13,35 ± 0,87	135,50 ± 5,61 0,40 ± 0,02 7,36 ± 0,37 54,00 ± 1,41 18,43 ± 0,55 340,33 ± 6,44 645,83 ± 124,34 5,90 ± 1,50 11,60 ± 0,33	136,17 ± 8,23 0,40 ± 0,03 7,18 ± 0,62 55,33 ± 1,51 19,02 ± 0,67 343,83 ± 5,71 647,83 ± 154,81 5,83 ± 0,51 11,18 ± 0,46

 

Table 12 and Table 13

Atlen SK.28-day repeated dose dermal toxicity study on rats.

Results of haematological investigation - males and females (satellite groups)

Study type	males of GROUP		females of GROUP
	0 SAT	1 SAT	0 SAT	1 SAT
HAEMOGLOBIN g/l HAEMATOCRIT 1/1 ERYTHROCYTES x 10^12/l MCV fl MCH pg MCHC g/l THROMBOCYTES x 10^9/l LEUKOCYTES x 10^9/l PROTHROMBIN TIME s	152,17 ± 3,87 0,46 ± 0,01 8,70 ± 0,24 52,83 ± 0,75 17,50 ± 0,37 331,50 ± 1,38 571,33 ± 136,55 7,30 ± 2,17 13,18 ± 0,69	148,83 ± 4,12 0,45 ± 0,01 8,33 ± 0,32 53,83 ± 1,17 17,87 ± 0,46 332,17 ± 4,40 601,33 ± 123,72 7,15 ± 1,79 12,93 ± 1,20	141,33 ± 10,25 0,40 ± 0,04 7,17 ± 0,85 55,67 ± 1,51 19,78 ± 1,10 356,00 ± 13,68 707,00 ± 266,61 4,67 ± 1,71 11,70 ± 0,36	139,83 ± 9,41 0,41 ± 0,03 7,49 ± 0,71 54,17 ± 1,17 18,73 ± 0,80 344,33 ± 9,00 505,00 ± 37,77 3,88 ± 1,60 11,20 ± 0,44

 

Table 14 and Table 15

Atlen SK.28-day repeated dose dermal toxicity study on rats.

Results of haematological investigation. Percentage content of leukocytes - leukocytogram – males and females

Study type	males of GROUP		females of GROUP
	0	1	0	1
NEUTROCYTES 1/1 EOSINOCYTES 1/1 BASOCYTES 1/1 LYMPHOCYTES 1/1 MONOCYTES 1/1 OTHER CELLS 1/1	0,19 ± 0,07 0,02 ± 0,04 0,00 ± 0,00 0,77 ± 0,09 0,01 ± 0,01 0,01 ± 0,01	0,14 ± 0,03 0,01 ± 0,01 0,00 ± 0,00 0,85 ± 0,04 0,01 ± 0,01 0,01 ± 0,01	0,12 ± 0,07 0,01 ± 0,02 0,00 ± 0,00 0,86 ± 0,07 0,00 ± 0,01 0,00 ± 0,00	0,18 ± 0,03 0,01 ± 0,01 0,00 ± 0,00 0,81 ± 0,03 0,01 ± 0,01 0,00 ± 0,00

 

Table 16 and Table 17

Atlen SK.28-day repeated dose dermal toxicity study on rats.

Results of haematological investigation. Percentage content of leukocytes - leukocytogram – males and females (satellite groups)

Study type	males of GROUP		females of GROUP
	0 SAT	1 SAT	0 SAT	1 SAT
NEUTROCYTES 1/1 EOSINOCYTES 1/1 BASOCYTES 1/1 LYMPHOCYTES 1/1 MONOCYTES 1/1 OTHER CELLS 1/1	0,25 ± 0,09 0,01 ± 0,00 0,00 ± 0,00 0,74 ± 0,10 0,01 ± 0,01 0,00 ± 0,00	0,26 ± 0,07 0,01 ± 0,01 0,00 ± 0,00 0,72 ± 0,08 0,01 ± 0,00 0,00 ± 0,00	0,18 ± 0,07 0,01 ± 0,01 0,00 ± 0,00 0,80 ± 0,06 0,01 ± 0,01 0,00 ± 0,00	0,17 ± 0,06 0,00 ± 0,00 0,00 ± 0,00 0,82 ± 0,07 0,01 ± 0,01 0,00 ± 0,00

 

Table 18 and Table 19

Atlen SK.28-day repeated dose dermal toxicity study on rats.

Results of bone marrow examination. Erythrocyte system – males and females

Study type	males of GROUP		females of GROUP
	0	1	0	1
PROERYTHROBLASTS 1/1 BASOPHILIC ERYTHROBLASTS 1/1 POLYCHROMATIC ERYTHROBLASTS 1/1 ORTHOCHROMATIC ERYTHROBLASTS 1/1	0,009 ± 0,007 0,049 ± 0,010 0,111 ± 0,009 0,117 ± 0,027	0,006 ± 0,005 0,041 ± 0,010 0,073 ± 0,024* 0,136 ± 0,017	0,006 ± 0,002 0,056 ± 0,024 0,092 ± 0,028 0,119 ± 0,029	0,005 ± 0,003 0,063 ± 0,025 0,109 ± 0,024 0,131 ± 0,031
TOTAL 1/1	0,286 ± 0,034	0,256 ± 0,036	0,273 ± 0,053	0,308 ± 0,028

* – statistically significant difference with p =< 0.05

 

Table 20 and Table 21

Atlen SK.28-day repeated dose dermal toxicity study on rats.

Results of bone marrow examination. Erythrocyte system – males and females (satellite groups)

Study type	males of GROUP		females of GROUP
	0 SAT	1 SAT	0 SAT	1 SAT
PROERYTHROBLASTS 1/1 BASOPHILIC ERYTHROBLASTS 1/1 POLYCHROMATIC ERYTHROBLASTS 1/1 ORTHOCHROMATIC ERYTHROBLASTS 1/1	0,006 ± 0,002 0,054 ± 0,014 0,075 ± 0,011 0,125 ± 0,019	0,005 ± 0,003 0,035 ± 0,012 0,098 ± 0,037 0,133 ± 0,022	0,011 ± 0,009 0,053 ± 0,013 0,099 ± 0,021 0,139 ± 0,015	0,009 ± 0,006 0,049 ± 0,029 0,097 ± 0,023 0,145 ± 0,030
TOTAL 1/1	0,261 ± 0,030	0,271 ± 0,061	0,303 ± 0,030	0,300 ± 0,057

 

Table 22 and Table 23

Atlen SK.28-day repeated dose dermal toxicity study on rats.

Results of bone marrow examination. Leukocyte system – males and females

Study type	males of GROUP		females of GROUP
	0	1	0	1
MYELOBLASTS 1/1 PROMYELOCYTES 1/1 NEUTROPHILIC MYELOCYTES 1/1 EOSINOPHILIC MYELOCYTES 1/1 NEUTROPHILIC METAMYELOCYTES 1/1 EOSINOPHILIC METAMYELOCYTES 1/1 BAND NEUTROPHILS 1/1 BAND EOSINOPHILS 1/1 SEGMENTED NEUTROPHILS 1/1 SEGMENTED EOSINOPHILS 1/1 SEGMENTED BASOPHILS 1/1	0,013 ± 0,007 0,069 ± 0,005 0,112 ± 0,017 0,022 ± 0,020 0,058 ± 0,013 0,015 ± 0,011 0,064 ± 0,021 0,007 ± 0,003 0,088 ± 0,014 0,004 ± 0,000 0,004 ± 0,000	0,009 ± 0,005 0,078 ± 0,022 0,113 ± 0,019 0,015 ± 0,005 0,063 ± 0,010 0,010 ± 0,003 0,074 ± 0,018 0,009 ± 0,006 0,130 ± 0,024* 0,004 ± 0,000 0,004 ± 0,000	0,009 ± 0,008 0,084 ± 0,015 0,096 ± 0,032 0,027 ± 0,013 0,065 ± 0,015 0,011 ± 0,008 0,049 ± 0,007 0,007 ± 0,004 0,101 ± 0,033 0,011 ± 0,008 0,004 ± 0,000	0,005 ± 0,002 0,062 ± 0,028 0,100 ± 0,017 0,024 ± 0,013 0,055 ± 0,017 0,015 ± 0,005 0,079 ± 0,038 0,009 ± 0,006 0,095 ± 0,026 0,010 ± 0,007 0,004 ± 0,000
TOTAL 1/1	0,455 ± 0,053	0,509 ± 0,045	0,465 ± 0,058	0,459 ± 0,034
Leukocyte system/erythrocyte system quantitative ratio	1,629 ± 0,381	2,044 ± 0,497	1,799 ± 0,640	1,505 ± 0,227

* – statistically significant difference with p =< 0.05

 

Table 24 and Table 25

Atlen SK.28-day repeated dose dermal toxicity study on rats.

Results of bone marrow examination. Leukocyte system – males and females (satellite groups)

Study type	males of GROUP		females of GROUP
	0 SAT	1 SAT	0 SAT	1 SAT
MYELOBLASTS 1/1 PROMYELOCYTES 1/1 NEUTROPHILIC MYELOCYTES 1/1 EOSINOPHILIC MYELOCYTES 1/1 NEUTROPHILIC METAMYELOCYTES 1/1 EOSINOPHILIC METAMYELOCYTES 1/1 BAND NEUTROPHILS 1/1 BAND EOSINOPHILS 1/1 SEGMENTED NEUTROPHILS 1/1 SEGMENTED EOSINOPHILS 1/1 SEGMENTED BASOPHILS 1/1	0,008 ± 0,005 0,074 ± 0,022 0,102 ± 0,013 0,027 ± 0,011 0,074 ± 0,021 0,016 ± 0,008 0,065 ± 0,026 0,009 ± 0,005 0,122 ± 0,046 0,009 ± 0,006 0,004 ± 0,000	0,006 ± 0,003 0,067 ± 0,019 0,103 ± 0,028 0,023 ± 0,011 0,075 ± 0,032 0,015 ± 0,007 0,093 ± 0,026 0,008 ± 0,004 0,117 ± 0,033 0,011 ± 0,006 0,004 ± 0,000	0,005 ± 0,002 0,070 ± 0,013 0,084 ± 0,019 0,029 ± 0,010 0,081 ± 0,020 0,019 ± 0,007 0,043 ± 0,021 0,006 ± 0,003 0,083 ± 0,030 0,019 ± 0,012 0,004 ± 0,000	0,004 ± 0,000 0,073 ± 0,016 0,109 ± 0,030 0,037 ± 0,013 0,073 ± 0,008 0,017 ± 0,011 0,051 ± 0,024 0,005 ± 0,002 0,093 ± 0,024 0,010 ± 0,006 0,004 ± 0,000
TOTAL 1/1	0,510 ± 0,059	0,520 ± 0,068	0,443 ± 0,048	0,475 ± 0,041
Leukocyte system/erythrocyte system quantitative ratio	1,999 ± 0,476	2,041 ± 0,674	1,487 ± 0,274	1,663 ± 0,499

 

Table 26 and Table 27

Atlen SK.28-day repeated dose dermal toxicity study on rats.

Results of bone marrow examination - different cells – males and females

Study type	males of GROUP		females of GROUP
	0	1	0	1
LYMPHOCYTES 1/1 MONOCYTES 1/1 PLASMOCYTES 1/1 MEGAKARYOCYTES 1/1 OTHER CELLS 1/1	0,129 ± 0,021 0,004 ± 0,000 0,006 ± 0,002 0,007 ± 0,003 0,112 ± 0,012	0,115 ± 0,025 0,004 ± 0,000 0,009 ± 0,006 0,009 ± 0,006 0,098 ± 0,009	0,133 ± 0,021 0,004 ± 0,000 0,008 ± 0,005 0,008 ± 0,005 0,109 ± 0,020	0,131 ± 0,030 0,004 ± 0,000 0,005 ± 0,002 0,006 ± 0,002 0,088 ± 0,011
TOTAL 1/1	0,259 ± 0,029	0,235 ± 0,024	0,262 ± 0,024	0,233 ± 0,028

 

Table 28 and Table 29

Atlen SK.28-day repeated dose dermal toxicity study on rats.

Results of bone marrow examination - different cells - males and females (satellite groups)

Study type	males of GROUP		females of GROUP
	0 SAT	1 SAT	0 SAT	1 SAT
LYMPHOCYTES 1/1 MONOCYTES 1/1 PLASMOCYTES 1/1 MEGAKARYOCYTES 1/1 OTHER CELLS 1/1	0,116 ± 0,019 0,004 ± 0,000 0,006 ± 0,002 0,006 ± 0,002 0,097 ± 0,027	0,110 ± 0,021 0,004 ± 0,000 0,004 ± 0,000 0,007 ± 0,004 0,084 ± 0,016	0,139 ± 0,022 0,004 ± 0,000 0,007 ± 0,003 0,007 ± 0,004 0,097 ± 0,022	0,108 ± 0,023 0,004 ± 0,000 0,005 ± 0,003 0,011 ± 0,009 0,096 ± 0,021
TOTAL 1/1	0,229 ± 0,041	0,209 ± 0,016	0,254 ± 0,024	0,225 ± 0,022

 

Conclusions:

Results of the 28-day repeated dose dermal toxicity study of Atlen SK on rats indicate that the dose of 1000 mg/kg b.w. can be considered a NOAEL for this endpoint.

Executive summary:

Presented results originate from a guideline study conducted in accordance with the requirements of the GLP. Hence, this information can be considered reliable and suitable for use as the key study for this endpoint.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Additional information

Atlen SK was tested in a 28-day repeated dose toxicity study (limit test) on rats at dose 1000 mg/kg bw/day administered via the dermal troute. No effects which could be related to the administration of the test item were found in the animals during the course of the study.

Oral administration of the Atlen SK for 90 consecutive days in the doses of 50, 125 and 300 mg/kg of body weight was well tolerated in all the animals and did not cause any signs of toxicity.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
One study available.

Justification for selection of repeated dose toxicity dermal - systemic effects endpoint:
One study available.

Justification for classification or non-classification

Data available for Atlen SK is conclusive but not sufficient for the classification of Atlen SK with regard to repeated dose toxicity.