Decan-1-ol, ethoxylated

Toxicity to reproduction

Administrative data

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 JUN 1982 - 22 JUL 1983

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1985

Report date:

1985

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Deviations:

yes

Remarks:

Only 3 applications per week; no or only partial examination of oestrous cycle and sperm parameters, no analytical purity reported

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Reference substance 001

EC Number:

614-482-0

Cas Number:

68439-46-3

Test animals

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Portage, Michigan, USA
- Age at study initiation: (P) 6 - 7 weeks; (F1) 4 - 7 weeks
- Weight at study initiation: (P) Males: 119.5 - 142.8 g; Females: 104.4 - 137.4 g; (F1) Males: 57.2 - 162.1 g; Females: 52.8 - 112.7 g
- Housing: animals were housed individually in wire-mesh bottom, polycarbonate cages
- Use of restrainers for preventing ingestion: no data
- Diet: Purina Formulab Chow (#5008), ad libitum
- Water: tap water, ad libitum
- Acclimation period: 19 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 1
- Humidity (%): 40 - 65
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 14 Jun 1982 To: 22 Jul 1983

Administration / exposure

Route of administration:

dermal

Vehicle:

water

Details on exposure:

TEST SITE
- Area of exposure: The test material was applied on the back of each animal.
- Time intervals for shavings or clippings: The back hair was shaved each week prior to initial weekly application and when necessary throughout the week to ensure adequate dermal exposure.

TEST MATERIAL
- Amount(s) applied: 1 mL/kg bw
- Concentration (if solution): 1, 10 and 25% (w/v)
- Constant volume or concentration used: yes

VEHICLE
- Amount(s) applied: 1 mL/kg bw

USE OF RESTRAINERS FOR PREVENTING INGESTION: no data

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: up to 3 weeks
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged (how): individually in a polycarbonate cage with Absorb-dri (nestwood chips) as nesting material

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Since dosing solutions were analysed by a combination of high performance liquid chromatographic method and gas chromatographic method, and were stable for at least 7 weeks, separate batches of dosing solutions were prepared approximately every four weeks. Rats were treated with a new batch of the appropriate dosing solution within 7 weeks. Each dosing solution was analysed prior to its use for verification of the test material´s concentration. The maximum observed deviation from the nominal concentration was 10%.

Duration of treatment / exposure:

(P) Males: 102 days before mating.
(P) Females: 170 days from the initiation of the study until the F1 generation was weaned
(F1) Males: 123 days at weaning, during growth into adulthood, mating and production of an F2 generation
(F1) Females: 221 days at weaning, during growth into adulthood, mating and production of an F2 generation, 30 days after last litter was weaned

Frequency of treatment:

3 days/week (except during mating)

Details on study schedule:

- Selection of parents from F1 generation when pups were 4 - 7 weeks of age.
- P: mating after119 days of dosing
- F1: mating after 133 days of dosing

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

10 mg/kg bw/day

Remarks:

nominal in water, corresponding to 1% (w/v)

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day

Remarks:

nominal in water, corresponding to 10% (w/v)

Doses / concentrations 3

Dose / conc.:

250 mg/kg bw/day

Remarks:

nominal in water, corresponding to 25% (w/v)

No. of animals per sex per dose:

30 P males, 30 P females
20 F1 males, 40 F1 females
16 F1 males (reversibility test)

Control animals:

yes, concurrent vehicle

Positive control:

Not applicable

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Observations were made once daily during treatment and mating periods for morbidity, mortality, general physical appearance, and clinical signs of toxicity. During gestation and lactation periods rats were observed in the morning and afternoon.

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: Male and female body weights were taken weekly up the time of mating. Females were weighed on days 0, 7, 14, and 20 of gestation and on days 1, 4, 7, 21 and 28 of lactation. Pups were weighed as a litter on days 1, 4, 7, 21 and 28 of lactation, and were also weighed individually on day 28 of lactation.

FOOD CONSUMPTION AND COMPOUND INTAKE: No

WATER CONSUMPTION AND COMPOUND INTAKE: No

Oestrous cyclicity (parental animals):

Not examined.

Sperm parameters (parental animals):

Parameters examined in P/F1 male parental generations: testes weight, sperm count in testes, measurement of lactate dehydrogenase isozyme (LDH-X) activity from testicular homogenates

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 10 pups/litter (5/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 / F2 offspring: number and sex of pups, stillbirths, live births, postnatal mortality, weight gain (litter), presence of gross anomalies, physical abnormalities

GROSS EXAMINATION OF DEAD PUPS:
- yes, for external and internal abnormalities

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals, as soon as possible after the last litters in each generation were produced
- Maternal animals: All surviving animals, after the last litter of each generation was weaned

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera

HISTOPATHOLOGY
The following tissues were prepared for microscopic examination: prostate, seminal vesicles, epididymides, right testis, both ovaries with oviducts, uterus with cervix

ORGAN WEIGHTS
- The following organs were weighed: right testes, ovaries with oviducts, uterus with cervix

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at ~28 days of age
- These animals were subjected to postmortem examinations (macroscopic and microscopic examination as follows: 5 pups per sex per dose

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera (F1 parental animals / F2 pups)

HISTOPATHOLOGY
- The following tissues were prepared for microscopic examination: stomach, duodenum, jejunum, ileum, colon, cecum, pancreas, mesenteric lymph nodes, cervical lymph nodes, liver, spleen, kidneys, urinary bladder, adrenals, lung, tongue, thyroids, larynx, trachea, esophagus, submaxillary salivary gland, heart, aorta, thymus, cerebrum, cerebellum, brainstem, pituitary gland, spinal cord, sciatic nerve, eyes, psoas muscle, skin, nasal turbinates, costochondral junction, uterus, cervix, ovaries with oviducts, seminal vesicles prostate, testes, epididymis.
ORGAN WEIGHTS
- The following organs were weighed: liver, kidney, lung with trachea and larynx, brain (including entire brainstem), spleen, testes, heart, ovaries with oviducts, uterus with cervix

Statistics:

Organ weight, organ weight/body weight ratio, LDH-X enzyme activity and litter size, sex ratio, survival index and gestational length were analysed by analysis of variance. Treatment groups were compared to the control group by one-tailed Dunnett´s t-test at p=0.05. Body weights were analysed by analysis of covariance. Body weights in all dose groups were adjusted for body weight on the first day of dosing due to differences in initial weights among the treatment groups. Gestational weights were adjusted to the common body weight on gestational day 0, and lactational weights were adjusted to that on lactational day 1. Pup weights and number of live and dead pups were adjusted for the differences in litter size among all dose groups. The analysis of covariance was used to adjust the treatment means for the differences in the covariates among the treatment groups. Subsequent treatment comparisons against the control were then made on the adjusted treatment means. The adjusted treatment means represent the means of the treatment groups if they were to have the same mean value in the covariate. Fertility indices were analysed by one-tailed Fisher´s exact test. Mating indices were analysed by Chi-square with continuity correction. Sperm counts and survival indices were analysed by non-parametric method, Kruskal-Wallis test. Treatment groups were compared to the control by one tailed Dunnett´s t-test at p=0.05 on the ranked data.

Reproductive indices:

Mating Index (%) = (No. of females mated / No. of females housed with males) x 100
Fertility Index (%) = (No. of females pregnant / No. of females mated) x 100

Offspring viability indices:

Survival Index (%) = (pups alive at day 1 / total born pups) x 100
Lactation Index (%) = (pups alive at day 28 / pups alive day 4 after culling) x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

F0 parental: 25% dose group: significantly decreased body weights; F1 males: 25% dose group: significantly decreased body weight at days 21, 28, 35 during dosing period (non-adverse)

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

F0 parental: 25% dose group: significantly decreased body weights; F1 males: 25% dose group: significantly decreased body weight at days 21, 28, 35 during dosing period (non-adverse)

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
Lacrimation, chromodacryorrhea, periocular swelling, ptosis, red nasal discharge, rhinorrhea, polyuria, soft stool, alopecia, odd-shaped pupil, and unkemptness were noted in the F0 and F1 parental rats in all dosage groups and the controls. In addition, the two highest dose groups, 10% and 25% of the test material, had a higher incidence of hunched posture. However, there were no pathological findings associated with these clinical signs. No mortality was observed in the F0 generation. However, five deaths were noted in the F1 generation. At necropsy, the posterior pharynx of each rat was filled with moist, finely ground and compacted food material that occluded the laryngeal opening of the rats. Similar material was also found in the esophagus of two rats and the trachea of one of these two rats. Death was apparently caused by asphyxiation and was not compound related because a similar finding was noted in the same strain of rats at another laboratory and a control rat was also affected.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
In the F0 adults, body weights of the 1% and 10% dose groups were not affected by the treatment. However, significantly lower (p<0.05) body weights of the 25% dose group were observed during certain intervals when compared to the control. There were statistically significantly lower body weights in the 25% dose group males between days 35-105 during the dosing period; in females, body weights were only significantly lower at day 49, but were slightly lower than the control during the rest of the dosing period. In the F1 adults, body weights of the 1% and 10% dose groups were not affected by the treatment when compared to the control group. However, body weights of the 25% dose group males were lower but only statistically significant (p<0.5) at days 21, 28 and 35 during the dosing period; body weights of the 25% dose group females were lower from days 14-119 during the dosing period when compared to the controls. There were no compound-related effects on maternal body weights of all dose groups during the gestational and lactational periods in the F0 and F1 generations when compared to the controls. On lactational day 14, maternal body weights of the 1%, 10% and 25% dose groups were statistically significantly higher than the control. These increases were of no toxicological significance.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
In the F0 parental males, testes weights in all dose groups were not affected by the treatment, whereas number of sperm per testes and per gram testes were significantly lower (p<0.05) in the 25% dose group when compared to the controls. Values of LDH-X enzyme activity in all dose groups were comparable to the control. However, in the F1 parental males, all the above mentioned parameters in all dose groups were comparable to the controls. The lower number of sperm in the testes of the 25% dose group F0 males was apparently due to a physiological variation as the values were within the normal range for this laboratory. Therefore, their difference is not considered to be a treatment-related effect. Since no effects were seen in F1 males, all rats prepared for the reversibility test were therefore killed and discarded.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
Mating indices were 73.3, 83.3, 83.3 and 93.3/ in the F0 generation, and 65.0, 70.0, 82.1 and 83.8% in the F1 generation of the 0, 1, 10 and 25% dose groups, respectively. The incidences appeared to be higher in the test material dose groups than the control in both generations but not significantly different from the control group by Chi-square test with continuity correction. The lower mating performance data from the control rats is commonly observed in this particular strain of rats. There were no apparent morphological differences in all rats to explain the apparent infertility. There were no compound related effects on fertility index and mean gestational length of the F0 and F1 females. The fertility indices were 77.3, 64.0, 76.0 and 53.6 in the F0 generation and 88.5, 67.9, 87.5 and 77.4% in the F1 generation of the 0, 1, 10 and 25% dose groups, respectively. Mated females were those having a copulatory plug or sperm in the vaginal smear or giving birth without the presence of copulatory plug or sperm. Mean gestational length in all dose groups in the F0 and F1 generations was approximately 22 days. Few females without the presence of copulatory plug or sperm during mating periods in both generations gave birth; their gestational length was therefore indeterminable.

ORGAN WEIGHTS (PARENTAL ANIMALS)
In the F1 adult males, significantly higher absolute and relative weights of the liver, lung and heart in the 25% dose group, higher absolute and relative weights of the lung in the 10% dose group, and higher relative lung weight in the 1% dose group were observed. The higher absolute and/or relative lung weight was not considered toxicologically significant as there was no dose-related linear relationship in all dose groups among the changes at the 1% and 10% dose levels. Also no morphologic changes were noted in the lungs. In the F1 adult females, significantly higher absolute and relative weights of liver, heart and kidney and the relative lung weight of the 25% dose group, higher absolute and relative heart weights and relative liver weights of the 10% dose group were noted.

GROSS PATHOLOGY (PARENTAL ANIMALS)
At necropsy, in the F0 generation, a significantly lower (p<0.05) carcass weight was observed only in the 25% dose group females, yet their absolute and relative organ weights were comparable to the controls. In the F1 generation, the carcass weight of the adult males and females were not affected.

HISTOPATHOLOGY (PARENTAL ANIMALS)
There were no significant differences in testicular histology from all test material treated rats and the controls in these two generations.

OTHER FINDINGS
According to the scoring table for skin reactions, individual skin irritation scores for all rats in all dose groups was zero throughout the F0 and F1 generations. The test material did not cause erythema or edema. However, both male and female rats in the 10% and 25% dose groups had dry and flaking skin. The discoloration of the application sites was noted in all treated rats including the controls. Possibly it was caused by the repeated shaving of the hair coat and dermal applications of the vehicle (water) or test solutions and was considered not to be compound related.

Effect levels (P0)

1

Dose descriptor:

NOAEL

Remarks:

reprotox

Effect level:

>= 250 mg/kg bw/day (nominal)

Based on:

test mat.

Remarks:

corresponding to 25% (w/v)

Sex:

male/female

Basis for effect level:

other: No treatment-related effects observed.

2

Dose descriptor:

NOEL

Remarks:

systemic

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Remarks:

corresponding to 10% (w/v)

Sex:

male/female

Basis for effect level:

other: No treatment-related effects observed.

3

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

>= 250 mg/kg bw/day (nominal)

Based on:

test mat.

Remarks:

corresponding to 25% (w/v)

Sex:

male/female

Basis for effect level:

other: Changes in body weight and organ weights without associated pathological findings.

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

Details on results (F1)

VIABILITY (OFFSPRING)
No compound-related effects on litter size, number of live pups and sex ratio of pups in the F1 and F2 generations were observed during lactational days 1, 4, 7, 14, 21 and 28. Litter sizes were 8.2, 9.6, 7.9 and 6.4 in the F1 generation and 6.9, 8.3, 8.0 and 8.4 in the F2 generation of the 0, 1, 10 and 25% dose groups, respectively. The mean litter size of the 25% dose group in the F1 generation was smaller than the control, but that of the control was the smallest in the F2 generation. However, there were no significant differences among the dose groups when compared to the controls. No compound-related effect on survival indices in all dose groups was noted. All pups dying during the pre-weaning period, including the control group, were examined grossly. Two pups, one male from a litter of the F1 10% dose group and one female from another litter of the F2 25% dose group were found dead, and they appeared to have been dehydrated. No pathologic finding was noted in these pups.

BODY WEIGHT (OFFSPRING)
There were no differences in body weights of the F1 and F2 pups in any dose group when compared to the control.

ORGAN WEIGHTS (OFFSPRING)
In the F1 pups, there were no differences in organ weights in any dose group when compared to the controls. In the F2 pups higher values of the absolute weights of liver, kidney, heart, spleen and ovary, and the lower relative brain weight were considered to be secondary to the higher carcass weights in this group (25% dose group) when compared to the controls. In addition to lack of dose response, there were no morphological differences among these affected organs when compared to the controls.

GROSS PATHOLOGY (OFFSPRING)
In the F1 pups, there were no differences in carcass weights in any dose group when compared to the controls. In the F2 pups, a significantly higher (p<0.05) carcass weight in the 25% dose group females was observed, but this increase was of no toxicological significance.

HISTOPATHOLOGY (OFFSPRING)
No treatment-related findings were recorded during histopathology, neither in the F1 pups nor in the F2 pups.

OTHER FINDINGS (OFFSPRING)
There was no compound-related effect on the development of the pups. Eye opening was observed at approximately 16.1-16.9 days for the F1 pups and 17.2-17.6 days postnatally for the F2 pups in all dose groups.

Effect levels (F1)

1

Dose descriptor:

NOAEL

Remarks:

reprotox

Generation:

F1

Effect level:

>= 250 mg/kg bw/day (nominal)

Based on:

test mat.

Remarks:

corresponding to 25% (w/v)

Sex:

male/female

Basis for effect level:

other: No treatment-related effects observed.

2

Dose descriptor:

NOEL

Remarks:

systemic

Generation:

F1

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Remarks:

corresponding to 10% (w/v)

Sex:

male/female

Basis for effect level:

other: No treatment-related effects observed.

3

Dose descriptor:

NOAEL

Remarks:

systemic

Generation:

F1

Effect level:

>= 250 mg/kg bw/day (nominal)

Based on:

test mat.

Remarks:

corresponding to 25% (w/v)

Sex:

male/female

Basis for effect level:

other: Changes in body weight and organ weights without associated pathological findings.

4

Dose descriptor:

NOAEL

Remarks:

dev tox

Generation:

F1

Effect level:

>= 250 mg/kg bw/day (nominal)

Based on:

test mat.

Remarks:

corresponding to 25% (w/v)

Sex:

male/female

Basis for effect level:

other: No treatment-related effects observed.

Results: F2 generation

Effect levels (F2)

Dose descriptor:

NOAEL

Remarks:

dev tox

Generation:

F2

Effect level:

>= 250 mg/kg bw/day (nominal)

Based on:

test mat.

Remarks:

corresponding to 25% (w/v)

Sex:

male/female

Basis for effect level:

other: No treatment-related effects observed.

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

Table 1: Animal assignment for mating

Dose Group	Number of rats and sex at start of study	Nominal value of test material in solution (% w/v)	Number of rats at scheduled necropsy
F0 Generation
A	30 M	0	30
	30 F	0	30
B	30 M	1	30
	30 F	1	30
C	30 M	10	30
	30 F	10	30
D	30 M	25	30
	30 F	25	30
F1 Generation
A	20 M	0	19 a
	40 F	0	40
B	20 M	1	20
	40 F	1	40
C	20 M	10	20
	40 F	10	39 a
D	20 M	25	19 a
	38 F	25	36 a

M: males

F: females

(a): deaths occurred before scheduled necropsy

Table 2: Reproductive parameters for F0, F1 and F2 generation

Parameter		Generation	Controls	1% (w/v)	10% (w/v)	25% (w/v)	Dose-response
Reproductive Performance
Mating index	%	F0	73.3	83.3	83.3	93.3	–
		F1	65.0	70.0	82.1	83.8	–
Fertility index	%	F0	77.3	64.0	84.0	53.6	–
		F1	88.5	67.9	87.5	77.4	–
Duration of pregnancy	Mean [days]	F0	22.2	22.1	22.2	22.3	–
		F1	21.8	22.1	22.0	22.0	–
Live birth index	%	F1	100	99.0	98.7	98.4	–
		F2	98.6	100	100	98.8	–
Litter size	Mean	F1	8.2	9.6	7.9	6.4	–
		F2	6.9	8.3	8.0	8.4	–
Pup weight (a) (day 1)	Mean [g]	F1	5.4	5.43	5.41	5.62	–
		F2	5.56	5.65	5.66	5.74	–
Pup weight (a) (day 28)	Mean [g]	F1	59.06	59.13	58.53	60.62	-
		F2	55.89	57.76	59.34	57.81	-
Sex ratio	% Males	F1	46	40	47	45	–
		F2	46	44	52	50	–
Survival index (day 1)	%	F1	100	98.6	98.9	97.8	–
		F2	98.4	99.6	98.3	97.3	–
Survival index (day 28)	%	F1	100	100	94.5	99.2	-
		F2	98.5	100	98.3	98.2	-
Lactation index	%	F1	100	100	98.6	100	–
		F2	98.5	98.8	97.4	97.4	–

(a): mean pup weights were calculated from litter weights divided by number of pups per litter

Table 3: Sperm characterisation for F0 and F1 males

Dose level in % (w/v)		Testes weight (g) (a)	Sperm/testes (million)	Sperm/g testes (million)	LDH-X activity (U/testes)
F0 Generation
0	Mean S.D: S.E: N	1.43 0.06 0.01 30	226.0 38.1 7.0 30	158.5 27.4 5.0 30	99.0 12.7 2.3 30
1	Mean S.D: S.E: N	1.42 0.05 0.01 30	211.2 23.8 4.4 30	148.6 16.6 3.0 30	97.2 12.8 2.3 30
10	Mean S.D: S.E: N	1.40 0.05 0.01 30	211.2 23.8 4.4 30	148.6 16.6 3.0 30	93.7 14.1 2.6 30
25	Mean S.D: S.E: N	1.42 0.06 0.01 30	204.7* 24.2 4.4 30	144.1* 17.5 3.2 30	97.0 11.1 2.0 30
F1 Generation
0	Mean S.D: S.E: N	1.40 0.09 0.02 19	247.6 36.4 8.3 19	178.1 28.8 6.6 19	113.18 7.19 1.65 19
1	Mean S.D: S.E: N	1.39 0.21 0.05 20	246.0 69.9 15.6 20	174.5 43.6 9.8 20	111.22 18.28 4.09 20
10	Mean S.D: S.E: N	1.41 0.08 0.02 20	250.3 40.3 9.0 20	17.7 29.7 6.6 20	114.57 7.03 1.57 20
25	Mean S.D: S.E: N	1.36 0.29 0.07 19	236.0 65.9 15.1 19	167.8 39.4 9.0 19	108.87 25.11 5.76 19

(a): detunicated

*: p ≤0.05 (one-tailed Dunnett´s t-test)

N: number of animals

S.D.: standard deviation

S.E.: standard error