Registration Dossier

Administrative data

Endpoint:
fertility, other
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1993
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other:
Remarks:
The endpoint addressed in this study, i.e. spermatogenesis in the male rat, does not cover completely all possible reasons for toxicity to reproduction. However, the given data indicate that the study was well-performed and meets scientific principles.

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
1993
Report date:
1993

Materials and methods

Test guideline
Qualifier:
no guideline available
Principles of method if other than guideline:
Male rats were injected intraperitoneally daily over 12 or 24 days with the test item. After sacrifice, testis were weighed, fixed and examined histopathologically for the stages of spermatogenesis.
GLP compliance:
not specified
Limit test:
yes

Test material

Constituent 1
Reference substance name:
Choline chloride
EC Number:
200-655-4
EC Name:
Choline chloride
Cas Number:
67-48-1
Molecular formula:
C5H14NO.Cl

Test animals

Species:
rat
Strain:
not specified
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: animal house of the Industrial Toxicology research Centre
- Age at study initiation: adult
- Weight at study initiation: 300 g
- Fasting period before study: no data
- Housing: in plastic cages in standard conditions of husbandry
- Use of restrainers for preventing ingestion (if dermal): not applicable
- Diet (e.g. ad libitum): standard animal feed (Lipton India) ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 1 week

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
water
Details on exposure:
Intraperitoneal injection
Details on mating procedure:
Not applicable
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
12 and 24 days
Frequency of treatment:
Daily
Details on study schedule:
Animals were divided into three groups: Groups I and II rats (n=25 each) were administered queous solution of choline chloride, 25 mg/rat intraperitoneally daily for 12 and 24 days. Group III (n=10) were administered distilled water only to serve as the control.
Animals of Group I were given ether anaesthesia and sacrificed on days 2,5,8,10 and 12 after 12 days of exposure. Group II animals were similarly sacrificed on days 2,5,8,10 and 12 after 24 days of exposure. Control rats were sacrified only on day 12 after both 12- and 24-day exposure schedules.
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
25 other: mg/rat/day
Remarks:
83 mg/kg bw/day (calculated assuming 0.3 g/rat)
No. of animals per sex per dose:
Treatment groups: 25 animals
Control group: 10 animals
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: 25 mg/rat intraperitoneally daily for 12 and 24 days. This dose was approx. 18% of the LD50 value of choline chloride in adult rats (450 mg/kg i.p).
In earlier studies three doses of choline chloride, i.e. 8, 25, and 40 mg/rat, were used. Because the 25 mg/rat produced moderate effects, it was selected for the present studies. further, based on the composition of the diet and average diet consumption per day/rat, 3 to 4 mg choline is ingested by a rat per day. Thus, the present dose gave a 6 to 8-fold excess availability of choline.
- Rationale for animal assignment (if not random): random
Positive control:
no data

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: No data

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: no data

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): not applicable

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): not applicable
Oestrous cyclicity (parental animals):
not applicable
Sperm parameters (parental animals):
Parameters examined in males: testis weight, epididymis weight, other: Histopathological analysis
Litter observations:
not applicable
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals after day 2,5,8,10,12 after exposure.

HISTOPATHOLOGY / ORGAN WEIGHTS
For the qualitative histopathologic analysis, individual stages were identified and the tubules were divided into stage groups, viz., I-IV, V-VI, VII-VIII, IX-XII, and XIII-XIV (according to Leblond CP, Clermont Y. Definition of the stages of the cycle of the seminiferous epithelium in the rat. Ann NY Acad Sci. 1952; 55:548-73). At least 20 tubules at each stage croup (total 100 tubules) per animal were evaluated at 400 x or 1000 x magnification to analyse specific effects on testicular tissues. This included peritubular membrane status, germinal epithelial status, cytoplasmatic vacuolation, cell sloughing, epithelial disorganization, cell death and cell type loss, presence of giant cells, spermatid damage, and inhibited spermiation.
The qualitative analysis, the quantitation of spermatogonia, zygotene, and pachytenes was performed in 10 randomly selected tubules at stage XII.
Organ weights of other tissues were determined (epididymis, liver, kidney, adrenal)
Postmortem examinations (offspring):
not applicable
Statistics:
Significance for changes in data was analysed by Student t test according Snedecor GE, Cochran WH. Statistical methods. Iowa City IA: Iowas state University Press; 1967
Reproductive indices:
not applicable
Offspring viability indices:
not applicable

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
not specified
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
No unscheduled deaths were noted.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
The average body weight gain in control animals between days 0 and 12 following both the experimental schedules was 20.00 + 2.3 g. The weight gain in treated animals was not significantly different from control values. No data on food consumption.
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not specified
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
The testis from all the animals of the control group exhibited normal histoarchitecture and active spermatogenesis.
Histopathological findings: neoplastic:
not specified
Other effects:
not specified

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
effects observed, treatment-related
Description (incidence and severity):
Treatment for 24 days increased stage XII spermatogenia count by day 5 posttreatment. The effects were reversible and contents of epithelial germ cells reached almost normal levels in the course of one seminiferous epithelial cycle.
Reproductive performance:
not examined

Details on results (P0)

REPRODUCTIVE FUNCTION: SPERM MEASURES / HISTOPATHOLOGY
12-Day choline chloride administration:
Animals administered Choline chloride for 12 days did not exhibit noticeable alteration in the organization of the germinal epithelium except on day 2, when epithelial vacuoles were seen at later stages. The nuclei of spermatogenic cells were pykontic at these stages but not at earlier stages. The presence of cellular debris in a few tubules suggested detachment of apical cytoplasm of Sertoli cells and sloughing of spermatogenic cells. By day 5, the testis recovered from initial injuries and the epithelium showed normal architecture through day 12. The quantitation analysis showed a partial effect on both spermatogonia and primary spermatocytes (Table 2).

24-Day choline chloride adminstration
Significant changes in testicular morphology were observed in rats exposed to Choline chloride for 24 days. Prominent features observed at day 2 included disorganization of the adluminal compartment of tubules mainly beyond stage VIII. Only a few tubules at stages I-IV were damaged. At stages V-VI epithelial vacuolation was observed. The tubules at stages IX-XIII were most damaged. Blebbing of Sertoli cell apical cytoplasm and dislodging of pachytene spermatocytes were marked at these stages. The arrangement of elongating spermatid bundles was inappropriate and part of the epithelium was devoid of elongating spermatids. In the tubules at earlier stages, at day 2, a decrease or absence of round spermatids was evidence of late pachytene degeneration earlier in time. The late pachytenes were highly eosinophilic. Sloughing of cells was found in only a few tubules in Group I as compared to those in Group II, At posttreatment day 5, spermatogonia and early primary spermatocytes in almost all the tubules were normal. Several pachytenes were necrotic and the adluminal portion of such tubules showed large gaps. At stages I-IV, the population of elongated spermatids was slightly decreased. At day 8, the tubules at stages XIII-XIV also showed gaps at the expected position of the elongating spermatids. These cells were thought to have been lost due to sloughing at earlier time intervals. By day 12, most of the tubules appeared to be regenerating and contained normal spermatogenic cells except a few necrotic pachytenes at stages XI-XII. The organization of the germinal epithelium appeared normal at earlier stages. The quantitation of spermatogenesis at stage XII showed an increase in spermatogonia at posttreatment days 5, 8, 10, and 12 (Table 2). Zygotenes were not altered in comparison to control counts. The most severe adverse effect was observed in pachytenes. Maximum depletion to 64% of control counts of these cells was noted at day 2. A marked recovery in cell counts towards normal was observed at day 12.

Effect levels (P0)

Key result
Dose descriptor:
LOEL
Effect level:
ca. 83 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
reproductive function (sperm measures)
Remarks on result:
other: recalculated from 25 mg/rat/day with a median body weight ranging from 300-320 g. based on mortality; body weight; organs weights; highest dose tested

Target system / organ toxicity (P0)

Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
83 mg/kg bw/day (actual dose received)
System:
male reproductive system
Organ:
germ cells
Treatment related:
not specified
Dose response relationship:
not specified
Relevant for humans:
not specified

Results: F1 generation

General toxicity (F1)

Clinical signs:
not specified
Dermal irritation (if dermal study):
not specified
Mortality / viability:
not specified
Body weight and weight changes:
not specified
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Sexual maturation:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
not specified
Histopathological findings:
not specified
Other effects:
not specified

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not specified

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not specified

Effect levels (F1)

Key result
Remarks on result:
not measured/tested

Overall reproductive toxicity

Key result
Reproductive effects observed:
no
Lowest effective dose / conc.:
83 mg/kg bw/day (actual dose received)
Treatment related:
no

Any other information on results incl. tables

Table 1. Testis weights of rats treated with Choline chloride

 

Posttreatment day

 

2

5

8

10

12

12(control)

Group I: 25 mg choline chloride/day for 12 days

Absolute weight (g)

1.18 ± 0.03

1.30 ± 0.60

1.41 ± 0.07

1.40 ± 0.16

1.48 ± 0.03

1.36 ± 0.09

Relative weight (g)

0.66 ± 0.04

0.56 ± 0.03

0.56 ± 0.03

0.51 ± 0.01

0.51 ± 0.01

0.71 ± 0.04

Group II: 25 mg choline chloride/day for 24 days

Absolute weight (g)

1.32 ± 0.13

1.51 ± 0.10

1.33 ± 0.05

1.41 ± 0.03

1.47 ± 0.07

1.55 ± 0.01

Relative weight (g)

0.53 ± 0.03

0.50 ± 0.01

0.48 ± 0.01

0.44 ± 0.01

0.51 ± 0.01

0.48 ± 0.01

The values are mean ± SE of 10 observations.

Table 2. Spermatogenic cell count at stage XII of the seminiferous epithelium following Choline chloride administration.

 

Posttreatment day

 

2

5

8

10

12

12(control)

Group I: 25 mg choline chloride/day for 12 days

Spermatogenia

43.23 ± 2.15

46.55 ± 3.08

50.47 ± 2.90

50.65 ± 3.50

51.12 ± 3.72

41.45 ± 2.05

Zygotenes

50.35 ± 3.68

54.02 ± 2.2

58.15 ± 2.52

62.35 ± 3.70

65.00 ± 2.55

57.35 ± 3.10

Pachytenes

65.55 ± 4.23

73.72 ± 3.50

75.50 ± 3.72

75.65 ± 3.15

71.75 ± 2.60

70.62 ± 3.00

Group II: 25 mg choline chloride/day for 24 days

Spermatogenia

50.25 ± 3.10

54.52 ± 2.68*

52.37 ± 2.60*

55.45 ± 3.35*

53.30 ± 3.57*

42.02 ± 2.28

Zygotenes

53.23 ± 3.65

53.27 ± 3.16

56.75 ± 3.88

52.40 ± 2.50

53.15 ± 3.27

56.23 ± 2.68

Pachytenes

45.25 ± 4.37*

50.25 ± 3.55*

58.60 ± 4.75

56.20 ± 3.15

62.44 ± 2.65

70.00 ± 3.20

Values are mean ± SE of 10 observations. *P<0.05.

Applicant's summary and conclusion

Conclusions:
The LOEL of 24 days of treatment with choline chloride was 83 mg/kg bw/day, based on spermatogenesis. Based on these data, the test item does not need to be considered toxic to reproduction.
Executive summary:

A study which evaluates the spermatogenesis in male rats after chloine chloride administration, has been included to cover the reproductive toxicity endpoint. Choline chloride was administered intraperitoneally to 25 adult male rats at dose levels of 0 and 25 mg/rat/day (ca. 83 mg/kg bw/day) for 12 or 24 days. The administration of the test item for 12 days did not significantly alter spermatogenesis and the treatment for 24 days had only transient effects. The LOEL of 24 days of treatment with choline chloride was 83 mg/kg bw/day, based on spermatogenesis. Based on these results, the test item does not need to be classified as toxic to reproduction according to CLP Regulation (EC) no.1272/2008.